Genetic Diversity Evaluation Of Cotton Germplasm And Cloning, Analysis Of Disease Resistance-related Genes To Verticillium Wilt

Quantification and classification of diversity in germplasm collection and utilization are important for both genetic researchers and plant breeders. Thirty transgenic bollgard cotton (Gossypium hirsutum L.) resources collected from different planting areas including 3 types of transformants were assessed by using simple sequence repeats (SSRs) and randomly amplified polymorphic DNAs (RAPDs). Jaccard's genetic similarity coefficients were calculated and dendrograms were constructed by the unweighted pair group method of arithmetic average (UPGMA) using the NTSYS-pc version 2.10. The result showed that all the resources could be divided into two main groups, but were not related to their origins. The genetic similarity coefficients were analyzed and found that the resources had relatively higher level in genetic diversity.Genetic diversity of 31 upland cotton cultivars was also assessed using RAPD and SSR markers in which 14 were introduced from USA and Iran. Totally 117 polymophic loci were obtained from 21 RAPD primers and 18 pairs of SSR primers. Jaccard's genetic similarity coefficients were calculated and dendrograms were constructed by the UPGMA method. Some of 17 Chinese cultivars and most of 14 foreign cultivars were grouped into two main clusters, while others were out of groups. That maybe infered there were more difference between these two kinds of germplasm. The similarity coefficient matrixes of the total 31 cultivars and the 17 cultivars from China were analyzed. The civil materials had higher level in genetic diversity compared with the materials from foreign counties. These results indicated that it was efficient to exchange the materials among different regions and introduce new genetic resources from foreign countries for enriching genetic constitution of local resources. And it is still important for germplasm to be introduced actively, evaluated properly and utilized effectively in our country.Verticillium wilt is a soil-borne fungal vascular diseases caused by Verticillium dalliae kleb., which has caused severe yield and quality losses in the upland cotton and other crops these years worldwide. The seaisland cotton {Gossypium barbadense) Pima 90 which believed to hold the resistant gene to Verticillium dalliae was used in this study. Roots were collected from the seedlings after inoculating pathogen with 2,4, 8,12,24,48, 72 and 96 hours and used for total RNA extraction with a modified simple protocol using guanidinium thiocyanate and chloroform-isoamylalcohol/phenol. The cDNAs from the inoculated seedlings acted as the tester and those from the uninfected seedlings were the driver. SSH method was employed to find the different expressed cDNAs responding to pathogen. T/A clone library was constructed which included 534 clones. The inserted cDNAs were amplied from the bacterial clones directly with M13 primers by PCR. The size of the products ranged from 0.2-1.2 kb with an average size of 0.5 kb. The SSH products were dotted on nylon filters, and the positive clones were screened by virtual Northern blotting with probes of the two kinds of initiative cDNAs. Totally 88 clones which expressed differently and had the potential to be involved in the defence response were identified and sequenced. Sequence similarity searches were performed with theBlastn and Blastx. Most of them showed high or part homology to genes or ESTs induced by different stresses in Arabidopsis thaliana and other species like the G hirsumtum. JSome disease resistance response-related genes like Pathogenesis-Related protein(PR)-10 family, disease resistance-responsive protein family and glutathione S-transferase (GST) were characterized. Some genes involved in transcription and signal transduction were identified like MYB family transcription factor, transducin family protein/WD-40 repeat family protein, Cys2/His2-type zinc finger protein etc. Two EST highly homologous to genes related to phytohormone regulator were also found. This would be helpful to understand the molecular mechanisms of disease response in cotton.Two genes GbPR 1 (Qossypium barbadense disease resistance protein-related 1) and GbPR 2 (Qossypium barbadense disease resistance protein-related 2) were isolated from the SSH library and characterized to be induced in the seedlings of Pima 90 upon inoculation with Verticillium dahliae. GbPR I contains a transcript of 701 bp, including a complete open reading frame (ORF) of 528 bp, which encodes a peptide of 176 amino acid residues, with a predicted molecular mass of 18.9 kDa and an isoelectric point of 6.34.The putative protein showed identities of 46% with a disease resistance responsive family protein of Arabidopsis thaliana, and shared a same conserved diligent domain. It exhibited a putative signal peptide cleavage site in its hydrophobic N-terminal part which was also just the same position following the part of the outside transmembrane helix predicated by TMHMM. And the TMHMM results also showed there is another transmembrane helix in its C-terminal part. ProtFun 2.2 predictions showed that it maybe play roles in the stress or immune response. Northern blotting and RT-PCR showed it is highly expressed during the inoculation with the Vertictilivm dalliae in sea-island cotton. GbPR 2 contains a transcript of 804 bp, including a complete ORF of 522 bp, which encodes a peptide of 174 amino acid residues, with a predicted molecular mass of 18.6 kDa and an isoelectric point of 6.59. The putative protein showed identities of 39% with the disease resistance responsive family protein of Arabidopsis thaliana, and shared the conserved diligent domain. Similar signal peptide cleavage site and function were found through bioinformatics analysis between GbPR 1 and GbPR 2. The most difference between GbPR I and GbPR 2 just located at N-terminal of putative proteins, no typical transmembrane helix was detected. MIC-3 considered highly induced by Meloidogyne was also found with high frequency in SSH library. It was also induced by the pathogen characterized by Northern blotting. And MIC-3 maybe a component involved in the basal stress response of cotton.The pathogenesis related (PR) -10 family was another gene family we characterized in the SSH library. PR-10 was found in many plants with ribonuclease-like activity in intro. It is considered playing an important role in plant basal response to biotic and abiotic stresses, though its function is still unknown. Three cDNA fragments with complete ORFs were isolated and the putative proteins encoded show significant homology to PR-10 proteins. Northern blotting and RT-PCR indicated they expressedconstitutely and also induced in cotton roots upon infection with pathogen. Southern blotting of sequences from Gossypium hirsutum, G barbadense, and G arboreum showed that PR-10 is a small, multigene family in Gossypium. All the 9 protein sequences of the PR-10 family from three cotton species in GeneBank are aligned based on Clustal X. The phylogenic tree divided the family into three subfamilies. Primers were designed for the N-terminus, a conserved motif GGPLGDKLEKI of the pathogenesis Bet v 1 domain, and the C-terminus of the proteins. RT-PCR results showed that the gene included one intron and two exons. PCR was employed to amplify in 14 materials representing 11 Gossypium species and 10 types of genome. The introns were rich in T and had a range of 74-98 nucleotides. Multiple alignments of the genomic sequences of PR-10 showed results similar to that of proteins. The exons and the introns within each subfamily were also highly conserved across species, highlighting this family's essential role in plant physiology. Also all the protein sequences with PR-10 conserved domain in plants were collected from Genebank. The phylogenic tree suggests that the evolution of the PR-10 family is parallel in different species in plants. Similar to R genes in plants the relatives of PR-10 in dicot and monocot are distant. One possible scenario is that the two paralogs of the PR-10 had diverged happened before monocots and dicots split. We suggest PR-10 proteins may have more important functions extending beyond adaptation to biotic or abiotic stress conditions.