Sugarcane - Bioenergy Crop Plants - page 537

method proposed by Wu et al. (2002). To do so, a software called OneMap, developed specifically
for this purpose (Margarido et al. 2007) was used. It can also be used for other outcrossing species.
Despite the superiority of this new analysis approach in relation to previous methods, in the case
of sugarcane, it is still possible to use markers with 1:1 and 3:1 segregations, which are known to be
less informative than other types (as for example, those that segregate 1:2:1 and 1:1:1:1 in the case of
diploid species). This makes it difficult to get linkage groups and also to order markers within these
groups. This leads to less saturated maps with less genome coverage. Furthermore, the integration
of maps of the genitors is not always possible.
The first sugarcane genetic maps were built using RAPD and RFLP type molecular markers.
Currently, mainly gene or genomic microsatellite molecular markers are used in the setting up of
genetic and molecular maps. Microsatellites or SSRs (simple sequence repeats) have become widely
used in plant marker studies. These markers are conventionally tandem repeats of small nucleotide
sequences of one to six bases in length. The variation in the number of repetitions results in poly-
morphic loci that are extremely useful, especially in mapping studies. The ability of SSRs to reveal
high allelic diversity is particularly useful in the discrimination between genotypes. The successful
use of this marker in other species such as barley (Saghai-Maroof et al. 1994; Russell et al. 1997),
rice (Wu and Tanskley 1993), wheat (Röder et al. 1995), apple (Szewc-McFadden et al. 1996), and
avocado (Lavi et al. 1994), stimulated the application of this technique to more genetically complex
species such as sugarcane (Cordeiro et al. 2000).
Nowadays, the search for SSRs is being carried out in expressed sequence tags (ESTs) deposited in
public databases, as this alternative is a simpler, faster and more economical strategy for the develop-
ment of SSRs. EST analysis is a simple strategy to study a portion of the expressed genome, even in
organisms with large, complex and highly redundant genomes, such as sugarcane. The basic strategy
for obtaining the EST is a fast and efficient method for genome sampling of gene active sequences. As
genetic markers, the EST-SSRs have been evaluated in several studies and tend to be considerably less
polymorphic than the markers generated from genomic sequences for rice (Cho et al. 2000), sugarcane
(Cordeiro et al. 2001; Pinto et al. 2006), wheat (Eujayl et al. 2002), and barley (Thiel et al. 2003).
The analysis in sugarcane of 8678 EST sequences revealed approximately 250 SSRs, the major-
ity made up of perfect trinucleotide repeats where (GCC) n , (CGT) n (CCT) n motifs were the
most common (Cordeiro et al. 2001). All selected EST-SSRs were polymorphic in the co-related
Erianthus and Sorghum genera. The lowest value for the polymorphic information content (PIC)
was obtained among the varieties of sugarcane (0.23), increasing between the species S. officinarum
and S. spontaneum (0.62) and reaching the highest value (0.80) among the genera Erianthus and
Sorghum . Because of the narrow genetic base of the varieties of sugarcane, the use of EST-SSR
can assist in the characterization of the genetic variability available in the germplasm collections of
related genera used in introgression programs. Thus, the introgression limitation of the Erianthus
genome in sugarcane ( Saccharum ) can be overcome by the use of EST-SSR in identifying the por-
tion of the Erianthus genome in intergeneric hybrids (Cordeiro et al. 2001).
The application of ESTs was shown to be a successful and efficient means of identifying sugar-
cane genes. A study by Carson and Botha (2000) showed that of all cDNA clones from the leaf iden-
tified in the search for homology, 38% showed significant similarity with known gene sequences.
This value can be compared with that observed in the analysis of cDNA libraries from the endo-
sperm and seed corn (39.3%, Shen et al. 1994) and even better than the results obtained using a
cDNA library from maize leaves (20%, Keith et al. 1993), tissues of different growth stages of rice
(25%, Yamamoto and Sasaki 1997) and portions of RNA from seeds, roots, leaves and inflores-
cences of Arabidopsis (32%, Newman et al. 1994).
21.7.2 t ranSgEnicS
Sugarcane biotechnology started in the 1960s with callus induction and rooted callus recovery
(Nickell 1964) followed by callus regeneration (Barba and Nickell 1969; Heinz and Mee 1969). The