ABSTRACT
Objective: To screen candidate genes involved in the terpenoid biosynthetic pathway of Tussilago farfara. Methods: The transcriptome of buds and leaves of wild T. farfara were respectively sequenced using the Illumina HiSeq 2500 high-throughput sequencing platform. The clean reads were de novo assembled by Trinity software, and the assembled sequences then followed by a series of bioinformatics analysis such as gene function annotation and differential expression gene. According to sequence annotation and differentially expressed genes analysis, the key enzyme genes related to the terpenoid biosynthesis were identified. Results: After high through-put sequencing, a total of 39 912 371 clean reads were obtained (SRA accession: SRR9113366, SRR9113367). The clean reads were then assembled into 91 118 unigenes. A total of 55 830 unigenes were annotated by a similarity search against NR, Swiss-Port, GO, COG, KEGG five public databases. Base on KEGG annotation and differentially expressed genes, totally 129 catalytic enzyme genes referring to the terpenoid biosynthesis were identified, including 91 terpenoid backbone biosynthesis genes, 32 terpene synthases, and 6 cytochrome P450 (CYP450) genes. Among them, 25 genes were differentially expressed. The expression of four enzyme genes in MVA pathway in leaves were higher than that in buds, while the five enzyme genes in MEP pathway were lower in leaves than that in buds. In addition, 10 genes were highly expressed in leaves, and nine genes were highly expressed in buds. According to the high expression of differentially expressed HMGR, TPS, AS, CYP450 genes in buds, it was speculated that these genes may be related to the high content of terpenoids in flower buds. Conclusion: This work obtained candidate key enzyme genes that may be involved in the biosynthesis of terpenoid by transcriptome sequencing. The results laid a foundation for further elucidating the molecular mechanism of terpenoid biosynthetic pathway in T. farfara.
ABSTRACT
Objective: The SSR loci information in the transcriptome of Tussilago farfara was analyzed and specific primers were designed, so as to provide powerful tools for molecular marker-assisted breeding in this plant. Methods: SSR loci in 18 938 unigenes with length of 1 kb or more obtained by transcriptome sequencing were searched by using MISA. SSR primers were designed by Primer3 and 55 pairs were randomly selected for the polymorphic analysis on 18 samples collected from different habitats. Results: A total of 4 688 SSRs were detected in the transcriptome of T. farfara, distributed in 3 844 unigenes with the distribution frequency of 24.75%. SSR loci occurred every 7 979 bp. Trinucleotide repeats appeared to be the most abundant SSRs with a frequency of 37.12%, followed by mononucleotide (32.36%) and dinucleotide (28.20%). Among all 60 repeat motifs, A/T (31.42%), AG/CT (12.80%), and ATC/ATG (9.62%) were the predominant repeat types. For validating the availability of the SSR primers, 55 pairs of primers were randomly selected for polymorphism analysis. Among them, 42 pairs (76.36%) produced clear and reproductive bands and 14 pairs showed polymorphism. Eighteen plants were divided into three groups by UPGMA. Conclusion: The SSR markers in the transcriptome of T. farfara show high frequency, rich type, and high polymorphism, which will provide the abundant candidate markers for genetic diversity, genetic mapping construction and marker-assisted breeding study for this plant.
ABSTRACT
In order to enrich the library of SSR and provide more powerful tools for molecular marker-assisted breeding in Astragalus membranaceus var. mongholicus, simple sequence repeats (SSR) loci in its transcriptome were searched in 18 040 unigenes (>=1 kb) by using MISA. SSR loci information was analyzed and SSR primers were designed by Primer 3. Furthermore, 110 pairs of primers were randomly selected for the polymorphic analysis on 20 plants collected from different habitats. A total of 5 640 SSRs were found in the transcriptome of A. membranaceus var. mongholicus, distributed in 4 462 unigenes with the distribution frequency of 31.26%. SSR loci occurred every 6 514 bp. Mono-nucleotide repeat was the main type, accounted for as much as 36.72% of all SSRs, followed by tri-nucleotide(32.57%) and di-nucleotide(27.73%) repeat motif. Among all 75 repeat types, A/T(2 026) was the predominant one followed by AG/CT(1 179), AAG/CTT(477). For validating the availability of the SSR primers designed using Primer 3, 110 pairs of primers were randomly selected for PCR amplification. Among them, 97 pairs of primers (88.18%) produced clear and reproductive bands. Using 19 pairs of primers showed polymorphism, 20 plants were divded into two groups by UPGMA. There are numerous SSRs in A. membranaceus var. mongholicus transcriptome with high frequency and various types, this will provide the abundant candidate molecular markers for genetic diversity, molecular identification, and marker-assisted breeding study for this plant.