In a previous study [10
], we sequenced 4800 ESTs from six normalized cDNA libraries of Asian seabass (Lates calcarifer
). From the 4800 ESTs, a total of 70 unique sequences containing microsatellites (repeat length: dinucleotide > 7, trinucleotide > 6, tetranucleotide > 5) from 130 clones were identified. Among the 70 microsatellites, 42 were CA-repeats, 23 GA-repeats, two GGA-repeats and three other types of repeats. These data indicate that unique microsatellite sequences accounted for 1.45% (70/4800) of cDNA clones in Asian seabass. CA- and GA-microsatellites were most abundant in cDNA of Asian seabass. However, they represent only 0.83% (40/4800) and 0.48% (23/4800) of cDNA clones from normalized cDNA libraries. Hence, straightforward random sequencing of clones from normalized cDNA libraries is not efficient for discovering microsatellites.
In this study, we tried to enrich CA-, GA-microsatellites from unnormalized cDNA of Asian seabass using biotinylated (CA)10
oligonucleotides, since these two types of microsatellites are most abundant in cDNA of Asian seabass. Two cDNA libraries were constructed, one enriched for CA-microsatellites and another for GA-microsatellites. From each library, 192 randomly picked clones were sequenced in both directions. Among the 192 clones from the cDNA library enriched for CA-repeats, 80 clones contained microsatellites. Of the 80 clones containing microsatellites, only 11 were singletons, and the remaining 69 were included in 8 clusters. A total of 19 (9.9%) unique microsatellites were obtained from the 192 sequences clones (Table ). Similarly, among the 192 sequenced clones from the cDNA library enriched for GA-repeats, 40 clones contained microsatellites. Eight were singletons, and 32 were included in 6 clusters. The cDNA sequence of the parvalbumin gene beta-1 containing one CT-microsatellites [10
] appeared 8 times in the 192 clones. A total of 14 (7.3%) unique microsatellites were obtained from the cDNA library enriched for GA-microsatellites. In comparison to the random sequencing of clones from normalized cDNA libraries without enrichment of microsatellites, the efficiency of microsatellite isolation from unnormalized cDNA libraries enriched for microsatellites has been raised over 10 times (for CA microsatellites: 9.9% vs. 0.83%; for GA-microsatellites: 7.3% vs.0.43%). In catfish, similar efficiency of isolation of microsatellites from cDNA was reported [18
]. However, high redundancy of cDNA sequences from unnormalized cDNA libraries reduced the efficiency of microsatellite isolation from cDNA.
Comparison of the efficiency of microsatellite enrichment from unnormalized and normalized cDNA libraries of Asian seabass
In order to increase the efficiency of enrichment of microsatellites, we tried to reduce the redundancy of cDNA by normalizing cDNA using duplex-specific nuclease (DSN) [20
] before enrichment of CA- and GA-microsatellites (Figure ). After cDNA normalization, redundant cDNA were removed (Figure ). Two normalized cDNA libraries, one enriched for CA-microsatellites and another for GA-repeats were created. From each library, 192 clones were sequenced in both ends respectively. Eighty-eight (45.8%) and 51 (26.5%) clones of the 192 clones from the normalized cDNA libraries enriched for CA- and GA-repeats respectively, contained microsatellites (Table ). The redundancy of clones was substantially reduced. In the 88 clones containing microsatellites from the cDNA library enriched for CA-microsatellites, 41 were singletons, the remaining 47 were included in 10 contigs. A total of 51 (26.5%) unique microsatellites were obtained from 192 sequenced clones. In the 51 clones containing microsatellites from the cDNA library enriched for GA-repeats, 35 were singletons, and 16 were included in 5 clusters. A total of 40 (20.8%) unique microsatellites we obtained from 192 sequenced clones (Table ). In comparison to the efficiency of microsatellite enrichment from unnormalized cDNA, the efficiency was about three folds increased by using normalized cDNA (for CA enrichment: 26.5% vs. 9.9%; for GA enrichment: 20.8% vs. 7.3%). Therefore, decreasing the prevalence of clones representing abundant transcripts before microsatellite enrichment by normalization of cDNA is essential for microsatellite isolation from cDNA. The normalization of cDNA using DSN was very simple and highly efficient in comparison to other cDNA normalization methods [21
]. The whole procedure of microsatellite enrichment starting from normalization of cDNA lasted only 5 days. Application of this method to isolate microsatellites from cDNA of grass carp brain got similar results (data no shown). Therefore the method is robust and reproducible.
Schematic presentation of the method for microsatellite enrichment from normalized cDNA. Details of each step can be found in the section "Methods".
Figure 2 Agarose gel electrophoresis (1%) of smart cDNA, normalized cDNA and cDNA enriched with microsatellites. Lane 1: 1 Kb ladder (NEB); Lane 2: smart cDNA; Lane 3: normalized cDNA; lane 4: 100 bp ladder (NEB); lane 5: normalized cDNA enriched for CA-microsatellites (more ...)
Sixty of 91 microsatellites isolated from the libraries enriched for CA- and GA-microsatellites had enough flanking regions for designing primers, and were characterized in a panel of 24 individuals previously used for characterization of microsatellites isolated from genomic DNA [22
]. Forty-one were polymorphic with an average allele number of 4.85 ± 0.54 ranging from 2 to 20 (Table ), whereas the average expected and observed heterozygosity were 0.56 ± 0.03 and 0.47 ± 0.04 respectively. The average allele number of microsatellites isolated from cDNA is slightly lower than those isolated from genomic DNA libraries, and characterized with same DNA panel [22
], which might be due to the relatively lower number of repeats of microsatellites identified from cDNA. Examination of genotyping errors using MicroChecker revealed no evidence for large-allele dropout or stutter-band scoring at any of the 41 loci. All 41 polymorphic microsatellites showed a Mendelian pattern of inheritance. Twenty-nine of 41 microsatellites were in HWE (Table ). Departure from HWE at 12 loci may be caused by the presence of null alleles. However, examination of genotypes using MicroChecker showed the possibility of presence of null alleles is low (P
> 0.05). Therefore, microsatellites isolated from cDNA using the described method could be useful for linkage mapping and comparative mapping and studies on genome evolution.
Characterization of 41 microsatellites isolated from cDNA of Asian seabass