Molecular cloning and characterization of cDNA-SSRs in Phalaenopsis orchids

• Main Authors: Shih-Yun Han, 韓世芸
• Other Authors: Wen-Luan Wu
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/17710032056860783023

碩士 === 國立成功大學 === 生命科學系碩博士班 === 94 === Microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are short (2-6 bp) tandemly repeated DNA sequences. Microsatellites are widely used as molecular markers in cultivar fingerprinting, genetic diversity assessment and marker-assisted selection because of their properties of genetic co-dominance, high reproducibility, multiallelic variation and high level of polymorphism as well as easily detectable by PCR. Microsatellites are present in both gene transcribed and nontranscribed regions. cDNA-SSRs may be associated with phenotypic traits and have higher transferability across related species. Phalaenopsis orchid is the most valued ornamentals and considered as an important floriculture industry in Taiwan. The development of very efficient SSR markers would be very useful for orchid cultivar identification and proprietary variety protection. The aim of this study was to isolate and characterize cDNA-SSRs for Phalaenopsis orchids. Firstly, the cDNA libraries enriched for microsatelltes of P. amabilis var. formosa and P. equestris were constructed and screened for AG/TC, AC/TG or AGG/TCC- microsatellite sequences. In total, 42 cDNA-SSR makers were obtained and characterized. Eight cDNA-SSR primer pairs were evaluated for amplification and genetic polymorphism in several P. amabilis or P. equestris accessions. The number of alleles per locus varied from 2 to 7 with a mean number of 4.1. The polymorphisms information content (PIC) ranged from 0.62 to 0.99 with an average of 0.85. The cross-species amplification of these cDNA-SSRs in 18 Phalaenopsis species was 76.4%, suggesting that the cDNA-SSR loci were highly conserved in Phalaenopsis. Sequences of the alleles at the locus PecAG002 from 18 Phalaenopsis species were used for phylogenetic analysis. The evolutionary pattern of cDNA-SSRs is consistent with that based on morphological characters. Positive amplifications at locus PecAG001 were obtained only in P. equestris, P. × intermedia and P. lindenii, indicating that this locus is species specific. Therefore, the locus PecAG001 was further analyzed on 30 commercial orchid accessions. This locus produced amplifications limited to accessions whose genetic contribution by P. equestris are above 3.22%. Moreover, the loci PecAG001 and PecAG002 were subjected to semi-automated fluorescent microsatellite analysis using ABI PRISMTM 310 genetic analyzer and accurate sizes of alleles were observed. Collectively, since these microsatellite makers developed in this study are primarily from expressed sequences, they can be used not only for variety identification but also for molecular breeding in orchids.