Identification And Cloning Of Fragments Linked To Spines In Carthamus Tinctorius Based On SRAP

The obtaining of significant agronomic trait gene linked markers will play an important role on developing molecular markers assisted breeding, raising the cultivar choosing efficiency and prediction, speeding the selection progress of new types. Carthamus tinctorius L. is 1-2 year herbaceous plant from Compositae or Asteraceae. The florets of Carthamus tinctorius L. are harvested for traditional chinese medicine for promoting blood circulation and removing obstruction in the channels. In China, outer involucral bract (OIB) without spines is an important agronomic trait in Carthamus tinctorius L. This is one of goals for choosing and cultivating new cultivar. As a result, identification the genes linked to spines of Carthamus tinctorius L. at the DNA or cDNA molecular level will lay a foundation for breeding spineless cultivars at the molecular level. Meanwhile, our study offers an effective way for the research of regulation mechanism of cultivar in Carthamus tinctorius L. at the molecular level.SRAP is a PCR-based marker system and aimed for the amplification of open reading frames (ORFs). It is based on two-primer amplification, since introns, promoters and spacers are usually variable among different individuals, this intrinsic dissimilarity makes it feasible to generate polymorphic bands based on introns and exons. It combines the advantagement of simplicity, reliability, high throughput ratio and facile to clone the selected fragments.Based on the strategy of bulk segregate analysis (BSA), two gene pools were respectively constructed according to the extreme trait of OIB with many long spines and no spines from F2 cross-generation safflower. Sequence-related amplified polymorphism (SRAP) method was employed to identify markers linked to spines in Carthamus tinctorius L. 45 pairs of SRAP primers were selected and screened from two gene pools, and one SRAP marker M3E3 was found to be linked to the spines in two parents and segregating F2 population confirmation. It was designated as M3E3. The result showed that extract length was 349bp. Furthermore, the cDNA-SRAP method was employed to identify markers linked to spines in Carthamus tinctorius L. There were 60 sets of primer combinations were selected and screened from two gene pools using reverse transcriptase-polymerase chain reaction (RT-PCR)-based cDNA-SRAP marker. Primer combinations were tested on cDNA from the two gene pools, of which, 19 primer pairs which revealed polymorphisms. Of the 19 primer combinations were tested from two gene pools and two parents, four primer combinations Me3/Em4, Me5/Em2, Me3/Em6, and Me2/Em1 produced fragment of SRAP-1, SRAP-2, SRAP-3, SRAP-4, SRAP-5 and SRAP-6 present in the spininess parent, spininess pool and absent in the spineless parent, spineless pool. To confirm the linkage of candidate SRAP markers to spininess, 40 spininess individuals and 30 spineless individuals were screened in the segregating F2 population and calculate crossover rates. High rates were found in SRAP-1, SRAP-2, SRAP-3 and SRAP-4 as 32.9%,45.7%,35.7% and 61.4% showing no linkage to spines. The crossover rate of SRAP-5 and SRAP-6 are 7.1% and 4.3% implying the fragments are linked to spines. We considered them specific fragments. Two bands at 278bp and 150bp were designated as GPY-1 and GPY-2.Based on the sequence data of the GPY-2, primers were designed for cloning of the full-length cDNA of GPY-2 using rapid amplification of cDNA ends (RACE) method. The full-length GPY-2 cDNA and the genomic sequence corresponding to the GPY-2 full-length cDNA were sequenced. The full-length cDNA sequence of Carthamus tinctorius L. was 1679 bp (GenBank Accession No: EF104641), consisting of a 80-bp 5'untranslated region, a 75-bp 3'untranslated region, and a 1524-bp ORF encoding a 508-amino-acid protein. Genomic sequence corresponding to the GPY-2 full-length cDNA was 1679 bp. The alignment result of GPY-2 cDNA sequence and the genomic sequence indicated that the GPY-2 gene intronless. Protein-protein BLAST showed that on the amino acid level GPY-2 protein shared high homology with ATP synthase CF1 alpha chain from other plant species. In our research, identification and use of PCR-based molecular markers linked to agronomically important genes in Carthamus tinctorius L. would enhance the effectiveness of the breeding program and provide experimental evidence for germplasm resources valuation and selection of Carthamus tinctorius L. Meanwhile, obtaining of the full-length gene linked to Carthamus tinctorius L. spines will lay a foundation for marker assisted selection (MAS) of Carthamus tinctorius L. at the molecular level.