International Journal of Seed Spices International J. Seed Spices 4(1), January 2014:88-90
Transferability of carrot (Daucus carota) microsatellite markers to cumin (Cuminum cyminum) Sushil Kumar*, Mahendi H. Asamadi, Ranbir S. Fougat, Amar A. Sakure and Jigar G. Mistry Department of Agri. Biotechnology, Anand Agricultural University, Anand - 388 110 (India) Practically no molecular tools have been developed so far for cumin (Cuminum cyminum) breeding. Microsatellite markers (SSRs) are becoming the markers of choice for marker assisted breeding, due to their high robustness and polymorphism. Due to their co-dominant and usually single-locus nature, SSR loci can be identified, and their alleles can be recognized in different varieties and genotypes of the same species and often in other closely related species also.However, the development of microsatellite markers through SSR library screening isrelatively a time consuming and expensive process (Yashoda et al., 9). SSR markers with good transferability can be applied for comparative mapping and genomic synteny among genera.Therefore, it is highly valuable to investigate the transferability of SSR markers among related species/genera.There have been several reports on the transferability of SSR markers in or across genera among different crops (Whankaew et al., 8; Fan et al., 4). Marker transferability offers a potential for low cost development of SSR markers for species with very little or no information on their genome. This can be accomplished through the screening of primers from different sources. But, the chances of successful crossspecies/genera SSR amplification are inversely related to the evolutionary distance between the two species. Under the present investigation SSR markers developed on carrot were tested for their transferability to cumin, both belonging to the Apiaceae (Umbelliferae) family, with an idea to enable the application of marker technology in cumin breeding.Two cumin lines (GC-4 and RZ-209) were used to evaluate the transferability of carrot SSR markers and one carrot line (GDC-1) was used as a positive control. DNA was isolated from seven days old seedlings using CTAB technique (Doyale and Doyale,3), purified and quantified using Nanodrop (Thermo scientific, USA). DNA was diluted to 20 ng/ul with TE buffer (10mM Tris–HCl, pH 8.0 and 0.1mM EDTA, pH8.0) and stored at 4ºC. A set of 50 SSR markers previously developed by Cavagnaro et al. (2) were analyzed. Initially, conditions used for SSR amplification were those described for the source species (carrot). For those SSR that failed to amplify or showed very weak amplification products, PCR conditions were optimized by increasing/increasing the annealing
temperature from 3 to 5ºC. PCR reactions were performed in a final volume of 15 µl containing 1x PCR buffer, 1µl MgCl2, 0.5µl dNTPs, 1µM of primers (10 pmol), 0.3µl of Taq DNA polymerase (5U/µl), and 20 ng of template DNA. Amplifications was carried out on thermal cycler(Eppendorf, Germany) under the following conditions: initial denaturation at 94ºC for 3 min, followed by 35 cycle of 94ºC for 30 sec, annealing temperature (Ta) recommended for the source-species amplification +5ºC for 40 sec, and 72ºC for 60 sec with a final extension of 10 min at 72ºC. The amplified fragments were separated on 1.5% agarose gel and documented using gel documentation system (Bio-Rad, Hercules, California). Experiment was repeated twice with each primer to examine the reproducibility. Amplification products were scored as positive only if a sharp and reproducible band of the expected size was observed. To evaluate the transferability of SSR markers, the microsatellite products were classified into two classes based on the band intensity and ease of scoring: (++) strong band and easy score and (+) weak band and difficult to score. The amplified fragments were classified as specific (+) if they produced amplification products with a size similar (within100 bp) to that of carrot (Table 1). In Apiaceae, only a few publicly available SSRs have been reported previously, and these were developed from carrot (9 SSRs [Niemannet al., 6]) and celery (11 SSRs [Acquadroet al.,1]). Given the advantages of identification of transferable markers in cumin, an economically important crop of Gujarat and Rajasthan, overall, 38% (19/50) of the tested carrot SSRs successfully amplified at least one PCR product of the approximate size expected (Table 1). Of these,12 primers (63%) showed a clear and strong PCR product (scored as ++). Amplified products were highly specific as15 primers showed a band of similar size to that of the carrot control (Table 1, Fig. 1). Few primers which could not amplify with the annealing temperature described for the SSRs of source species, the annealing temperature was changed to + 5°C. Modifications in the PCR protocols especially annealing temperature successfully amplified the product. Such change is recommended earlier also for the successful transfer of microsatellite primer pairs between species 88
*Corresponding author: E-mail:
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International Journal of Seed Spices
(Lendvay,5). Rossetto (7) reviewed cross-species transferability in several plant species and concluded that the average transferability across species in the same genus was 76.4%, and across related genera was 35.2%.The outcomeof present study indicatethat there is a potential for transferring SSR markers ofcarrotincumin. These results are in agreement with Cavagnaro et al. (2) where transferability of SSRs across Apiaceae taxa was reported to widely vary among the accessions and was
found highly associated with the accessions’ phylogenetic relatedness to carrot. Transferability of carrot SSRs across genera has confirmed in this study which indicate that the sequences flanking the microsatellite regions are conserved between carrot and cumin. Overall, results suggest that there is good scope for wide use of identified markers in population genetic diversity, molecular breeding and evolutionary studies in cumin and also in among other Apiaceae species.
Table 1.Transferability of carrot microsatellite markers to cumin. Sr. No. 1
Primer
Primer Sequence
Tm (°C)
GSSR3
Product Size (bp)
Quality1
Specificity2
TTCTTCTTCATCTCTCCACAAGG/ 50 266 ++ + TAAAACAGTCACACATCTCTC 2 GSSR5 ATAATAAACCCAACCAGACCCC/ 54 235 ++ ATCAGGCAAATCCCATACTGAC 3 GSSR35 AATTCACAATCACCGACTCTCC/ 54 314 ++ ACGTCAAAGCTCCTGTTCATTT 4 GSSR81 TGGGTCTCTGGCCAATTCTA/ 52 233 + + CAATTTGCACATAACTCATCAAG 5 GSSR96 AGCGTCGTTTTCGCGAGT/ 51 357 ++ + CGCGGTTAAAGCAAAGCTAAT 6 GSSR111 GAGGAAGGGTAGATCCAGTCA/ 59 295 ++ + ATGGGATGTCTTTCCCCTCTAT 7 GSSR113 AGTGGTTGTGAGGTTGATTGTG/ 52 346 + + TATGTCGGAAAGGTTCAATGCT 8 GSSR140 GGATACGAAGGAAAGACTCCAC/ 52 484 + AGGAGAGTAAAAGATTGAGGACTTG 9 GSSR142 GTAGAAACCTTTTGGCAGTAACG/ 54 108 + + CCAAGACCATGAAGAAATCACTC 10 BSSR14 TACCCATAACTCAAGTTGGATAATTC/ 54 224 ++ + AATGTCTAAACCCACTGATTTAAAAG 11 BSSR24 AACTGACCAGTTAAAATCTCCAGTAT/ 59 212 ++ + TCACTTAAAAGTCTACAAAATGTGCT 12 BSSR25 GTTGTTCCTATTCAGAGGACTTGT/ 52 266 ++ + GGTAGTCTTGGAGGAGTTGAAGT 13 BSSR40 AAACAACCAACGTCTAGCCTAAGTTC/ 54 228 + + CATATTAACGAGGTCCGGACTAATCT 14 BSSR41 AGCAACAGTAGCTTCCTTGATCTGTA/ 56 172 + AACATCACATCTGGATCTAAGCAAAG 15 BSSR65 GGAAAAATTGCACCAGTAGTTGAAGCT 60 216 ++ + / CGGTCTGTTTAAGGTGACAAAACTTG 16 BSSR86 CCTAATTCGAGCCAACTTTAA/ 56 233 ++ + TTGGGCTCGATTAAGTGTAAAAT 17 BSSR87 TAAATGTGAATTCTCAAATGTCTCG/ 56 212 & 322 ++ + GAGTTAGTGTAAATTGGGGCTTTTT 18 BSSR88 CTGAATTTGGATAAGAATTTCAAGTG/ 55 189 ++ + TGGTATGAAACCAAAATACGC 19 BSSR96 AATGGTGTGAAGATTGTGCTTCTT/ 51 200 + + CTACAAATGTTCAATGCTCCTCTTC 1 ++= strong band and easy to score; + = weak band and difficult to score 2 + = amplified product of a similar size (within 100 bp) to that of carrot; - = amplified product was not of a similar size
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Fig. 1 A gel image showing carrot SSR markers amplification patterns in cumin (GC-4)
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Received : August 2013; Revised : October 2013; Accepted : December 2013.
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