Characteristics Of The Functional Genome And Repetitive Sequences In Porphyra Yezoensis

Porphyra yezoensis Ueda is a genus of red macroalga considered to be of important economic and research value. It is also an ideal model species for studying on the stress-tolerance molecular physiology of intertidal seaweeds. However, the knowledge of their environment-tolerance mechanisms and molecular breeding are very lacking at present. In this thesis, comprehensive and systematic study of functional genomics and repetitive sequences will substantially improve the global view of the P. yezoensis transcriptome and its genome structure, and will be beneficial to improve the stress-tolerance breeding and cultivation techniques.Illumina/Solexa sequencing technology is used to study the transcriptome and small RNA of P. yezoensis with the aim to characterize its functional genome. Comparative analysis of the composition and distribution of repetitive sequences is performed in P. yezoensis. A group of microsatellite DNA markers are developed and used to determine the pure line and genetic diversity in Porphyra.The following are the major findings:The transcriptome of P. yezoensis was profiled with next-generation RNA sequencing technology. A total of 13,333,334 quality paired-end reads were obtained from the cDNA library constructed for a mixture of P. yezoensis at different growth stages and under different growing conditions, which generated 1.2×10~9 nt. The assembling of these reads yielded 31,538 unigenes with a mean length of 419 nt. The unigenes obtained overlapped the majority of the deposited in dbEST database at NCBI. A large set of annotated genes involved in stress tolerance and defense, and a C4-like carbon fixing pathway was profiled. Less than half of them can be annotated based on the similarity with the known proteins in diverse databases, while the remaining should be novel.Deep sequencing technology is used and 8,262,593 quality small RNAs are obtained from a small RNA library. The largest number of small RNAs are length of 22 nt. By blasting small RNAs cloned more than once (682,398) against known databases, all small RNAs were classified into different categories, including rRNA, tRNA, snRNA, snoRNA and unannotated RNAs. By blasting against miRBase, 3 small RNAs were found to be the same as plant miRNAs. Eight novel miRNA candidates were predicted from the GSS of P. yezoensis as well as from the sequencing results. By prediction, six novel miRNA candidates result in ten target genes, most of which encode proteins involved in metabolism, transportation and signal transduction.Comparative analysis of repetitive sequences show their characteristics of composition and distribution in P. yezoensis. The total length of minisatellite repeats in P. yezoensis is less than 2% of total sequences analized with low repeat times. Most of the minisatellite DNA is composed of four types of bases with GC-rich. The repeat times, number and length proportion of minisatellite repeats in genome sequences are lower than those in transcribed sequences. The total length of microsatellite repeats in P. yezoensis is less than 0.4% of total sequences analized. The repeat times of most microsatellite repeats are less than nine. Trinucleotide repeat types are the most abundant, and CCG is the most abundant motif. The frequency of microsatellite repeats in genome sequences is lower than that in transcribed sequences, whereas, the length proportion is higher. In P. yezoensis, RNA transposons are much more than DNA transposons. The most abundant type of RNA transposons is the LTR retrotransposons, followed by LINE elements and SINE elements. Among all elements, gypsy made up the largest fraction in number. The frequency and length proportion of transposable elements in genome sequences are higher than those in transcribed sequences. In short, the length proportion of repetitive sequences in genome is approximately 6.5%, which is much higher than that in transcribed sequences. However, the frequency of repetitive sequences in genome is lower than that in transcribed sequencesTwenty-four microsatellite DNA markers are developed from enriched library of P. yezoensis. Twelve primer pairs amplify two to four bands, whereas another 12 primer pairs produce monomorphic banding patterns. A total of 29 alleles are produced at 12 loci, with an average of 2.42 alleles (Na) and 1.81 effective alleles (Ne) per locus. These markers are used to analyze the genetic diversity within 11 geographically different lines of P. yezoensis. Overall, these lines are clustered into two divisions with those from close geographic locations clustering together. Further cloning and sequencing of size variant alleles at two microsatellite loci from one blade reveals that the variable numbers of motif repeats in different alleles are major sources of polymorphisms. In P. haitanensis, nine and fourteen microsatellite DNA markers are developed from EST and enriched library, respectively. These markers are also used to determine pure line and genetic diversity of P. haitanensis, which proved their value of practical applications.