Identification of new microsatellite DNA markers for sugar and related traits in sugarcane

Abstract

Molecular studies in sugarcane are relatively limited because of the complex genetic structure, long life cycle and non availability of reliable sugarcane specific molecular markers. The use of DNA markers for the genetic analysis and manipulation of desirable agronomic traits has become an increasingly useful tool in sugarcane breeding. The objective of the present study was to evaluate the polymorphic potential of sugarcane microsatellite markers developed at National Research Centre on Plant Biotechnology, IARI, New Delhi in high and low sugar lines. These microsatellites markers were screened and validated for their polymorphic, genetic diversity, cross-transferability and comparative linkage mapping potential in high and low sugar bulk of two segregating progenies and 20 each, cultivated high and low sugar commercial varieties. One hundred sixty eight (28%) of the microsatellite markers were found to be highly robust and polymorphic with PIC values ranging from 0.51% to 0.84%. Forty three (0.26 %) markers contained dinucleotide repeats, fifty seven (0.34 %) markers contained trinucleotide repeats, twenty five (0.15 %) and forty three (0.26 %) markers contained composite repeats. The number of observed allele ranged from 2 to 11, with an average of five alleles detected per locus. A total of 977 polymorphic DNA bands were identified, with their fragment size ranging from 20 to 1380 bp. These microsatellite markers are an ideal resource for use in managing sugarcane germplasm, trait mapping and marker assisted breeding strategies. The wide cross -species transportability of these markers may extend their value to research involving other Saccharum species complex.

Use of differential levels of mean observed heterozygosity in microsatellite loci of commercial varieties of sugarcane (Saccharum spp)

“…The mean number of alleles estimated in our study (4.12 alleles/locus) was also approximately consistent with the mean number of alleles reported in other RB sugarcane varieties (3.2 alleles/locus) (Duarte-Filho et al, 2010), indicating the great potential and high reproducibility of these microsatellite loci to investigate genetic divergence in the sugarcane germplasm. Using a higher number of SSR primers, other breeding programs have reported a correspondingly higher number of alleles at SSR loci (Singh et al, 2008(Singh et al, , 2011Oliveira et al, 2009;Liu et al, 2011).…”

“…The analysis of SSR loci in sugarcane has principally been scored based on the genetic distances, where the estimation of the effective number of alleles and the H O were not calculated (Singh et al, 2008(Singh et al, , 2011Oliveira et al, 2009;Duarte-Filho et al, 2010;Liu et al, 2011;Silva et al, 2012). In our study, the highest heterozygosity detected at SSR loci, e.g., in the RB72454 variety, is potentially meaningful because high heterozygosity would indicate that the plant population likely has a substantial amount of adaptive genetic variation to escape the effects of a control agent that limits the development and maintenance of the plants ( Allendorf and Luikart, 2007), in contrast to plant populations showing a lower level of heterozygosity.…”

“…The 40 SSR primers previously mapped for sugarcane by the International Sugarcane Microsatellite Consortium (Cordeiro et al, 2000;Singh et al, 2008;Oliveira et al, 2009) were synthesized by Invitrogen Technologies Corporation (USA) and used with 5 DNA samples from each sugarcane variety. After screening the 40 SSR primers, a set of 17 SSR primers (Table 2) was selected for further analysis.…”

“…The amplifications were performed by the Touchdown (Td) PCR program (Don et al, 1991) using a primer-specific annealing temperature (TA). Cordeiro et al (2000); Singh et al (2008); Oliveira et al (2009). TD = Touchdown (Don et al, 1991).…”

“…Considerable advances have been made in the area of sugarcane genetics through the use of microsatellite markers. The associations of markers with sugarcane diseases (Wei et al, 2006) and sugar contents (Singh et al, 2008(Singh et al, , 2011 have been investigated using sugarcane clones. Microsatellite markers have many desirable genetic attributes, including hypervariability, wide genomic distribution, co-dominant inheritance, reproducibility, multi-allelic nature, and chromosome-specific location.…”