Submitted to: Euphytica
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/26/2005
Publication Date: 10/15/2005
Citation: Hu, J., Ochoa, O.E., Truco, M.J., Vick, B.A. 2005. Application of TRAP technique to lettuce (Lactuca sativa L.) genotyping. Euphytica. 144:225-235. Interpretive Summary: Using the newly developed TRAP (target region amplification polymorphism) technique, we identified DNA markers that could be used to analyze genetic variation of a germplasm collection containing 53 lettuce cultivars and six accessions of two wild lettuce species. The studies were done in collaboration with researchers from the University of California at Davis. With the TRAP method, informative DNA markers were generated by PCR reactions using primers that were designed from DNA sequences published in the Compositae EST (expressed sequence tag) database housed at UC Davis. Selection of the ESTs for primer designing from the database was based on their homology to the known genes involved in either governing development or conferring resistance to diseases. From 10 sets of PCR reactions we were able to identify 107 markers that were useful in distinguishing DNA differences among the cultivated lettuce accessions, and revealed the evolutionary relationship among the three species. Specifically, the cultivated species L. sativa is more closely related to L. serriola than to L. saligna, which is in accordance with the previous results obtained with RFLP and isozyme markers. In addition, the markers could be used to distinguish all the cultivars. Four types of cultivars with different horticultural characters were used in the current study. Using statistical cluster analysis, the cultivars could be grouped according to horticultural types with a few exceptions. The markers were also used to estimate the genetic variability among the cultivars within each horticultural type. We found that the butterhead type has the highest genetic diversity and the romaine type possesses the lowest genetic diversity, while the iceberg and leaf types have intermediate genetic variability. Most of the TRAP markers segregated according to the expected genetic ratio in a recombinant inbred line population and are ready to be added into the existing lettuce linkage map. The result will be useful for the genetic improvement of lettuce.
Technical Abstract: The newly developed, PCR-based TRAP (Target Region Amplification Polymorphism) technique has been applied to evaluate genetic variability among lettuce (Lactuca sativa) cultivars and wild relatives. Fifty-three cultivars and three accessions from each of the two wild species (L. saligna and L. serriola) were used in the current study. The fixed PCR primers were designed against the four EST sequences homologous to the homeobox genes, to one leucine rich repeat, and to one unique sunflower sequence. In combination with four fluorescent labeled arbitrary primers, 10 PCR reactions amplified 769 fragments of 50 to 900 bp in length. Scoring of the fragments showed that 388 were polymorphic among the 59 Lactuca entries, with 107 fragments being polymorphic among the 53 lettuce cultivars and the six accessions from two wild species. There were 251 fragments that were present only in the wild species. These markers not only discriminated all cultivars but also revealed the evolutionary relationship among the three species: L. sativa, the cultivated species, is more closely related to L. serriola than to L. saligna. Cluster analysis grouped the cultivars by horticultural types with a few exceptions. These results are consistent with previous findings with RFLP, AFLP, and SSMPL markers. These TRAP markers also revealed significant differences in genetic variability among horticultural types, measured by the average genetic similarity among the cultivars of the same type. The leaf type and butterhead type possess relatively high genetic variability, at 58.3% and 60.1%, respectively; the iceberg type has moderate variability of 70.3% and the romaine type has the lowest variability at only 82.9%. Most of the polymorphic markers segregated in the expected Mendelian ratio in the permanent mapping population derived from an interspecific cross between L. sativa and L. serriola and could be easily integrated into the existing lettuce linkage map.