DNA sequence analysis of the SSR markers for hybridity of transgenic F1 hybrid papaya with delayed ripening trait and papaya ringspot virus resistance

Date

4-2013

Degree

Bachelor of Science in Agricultural Biotechnology

College

College of Agriculture and Food Science (CAFS)

Adviser/Committee Chair

Roberta N. Garcia

Abstract

Abstract: Carica papaya L. production is greatly affected by the occurrence of papaya ringspot virus and post-harvest losses. To address these two major problems, a hybrid between an elite BC3 line with PRSV resistance and a transgenic papaya with delayed ripening trait was developed. Furthermore, SSR markers using CPY primers designed from papain (M15203) were generated to ascertain the hybridity of the F1. In this study, the use of SSR markers in hybridity testing of F1 hybrid from each parental line was validated through DNA sequence analysis. Genomic DNA of the transgenic line, F1, and BC3 were subjected to PCR for amplification of target sequences. Visual analysis of the band intensity of PCR products showed that F1 hybrid had more intense band of 950 bp, 600 bp and 550 bp, and is thought to be inherited from both parental lines in an additive manner. The multiple sequence alignment of the 550 bp amplicon showed high percent similarity (86.0% between F1 and T4, and 51.0% between F1 and BC3) in certain locations, which implied that the conserved sequences of both parents contribute to ix functional roles. A similar result was observed in sequence alignments of the 600 bp and the 950 bp PCR products. Moreover, sequence alignments with papain showed high percent similarities with the 600 bp and 550 bp amplicons. For the 550 bp amplicons, the obtained percent similarity values with papain were 70%, 74% and 67% for F1, T4 and BC3, respectively. On the other hand, the 600 bp amplicons of F1 and T4 had 72% and 68% nucleotide sequence similarity with papain. The tandem repeats found in the 550 bp amplicon were ATAT, TATA, AGAG, ATAT and TCTC. The 600 bp amplicon showed AGAG, ATAT, TCTC, TATA tandem repeats while TATA, TCTC, ATAT, AGAG, AGAGAG were found in 950 bp amplicon. Thus, this study confirmed that this CPY SSR marker system is a robust molecular marker that could distinctly identify the hybrid nature of the transgenic F1 as evidenced by the nucleotide sequence of the generated PCR products.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 2013 A127 /Y66

Document Type

Thesis

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