PCR Detection And In Situ PCR Location Of Specific DNA From Sugarcane Parental Species

Modern sugarcane cultivars(Saccharum spp.)are polyploid or aneuploid clones of complex genetic background, with major components of the genome derived from S. officinarum, and the remainder from S. spontaneum, S. barneri, S. robustum, S. sinense, and Erianthus arundinaceus. In this thesis, a series of species/genus-specific DNA fragments of parental sugarcane were screened out by RAPD and ISSR markers and then cloned. Based on the cloned specific DNA fragments, PCR markers were developed. SSR primers for detection of species/genus-specific DNA were also selected. Then the genetic dynamic states of sugarcane parental specific DNA were analyzed between parents and their progenies. A few of species/genus-specific DNA sequences were located on chromosomes by in situ PCR. And the genetic base of sugarcane germplasm was analysed by ISSR marker.The technique system which could identify the structural character of species genuis component from parent in sugarcane genome rapidly and accurately was developed. These results could be the new bases and method for the research of sugarcane genetic analysis, species identification, parental selection for hybridization and so on.113 bands of specific DNA fragments were screened out, including 53 specific bands of S. spontaneum, 19 bands of S. officinarum, 10 bands of wild species of Saccharum spp. and 31 polymorphic bands of parent. Integrated with 46 bands of sugarcane parents which were generated by AFLP but still not purified, there were 159 bands of specific DNA fragments. The sequences of 72 bands of specific DNA fragments were sequenced and the actual sequence compositions of 68 bands were obtained.Based on that 122 pairs of primers were designed and synthesized, 4 types of specific PCR primers were screened out by using 12 sugarcane parental genomes as templates, including 29 pairs of S. spontaneum, 14 pairs of wild species of Saccharum spp.,27 pairs of Saccharum spp.and 2 pairs of Erianthus arundinaceus.With the genomic DNA of 39 sugarcane parents and 57 cultivars were amplified with some specific PCR primers, the dynamic and transferability of sugarcane parental specific genome were analyzed. The results showed that the specific DNA fragments could recover in cultivars with different probability. Among the recovering probability of 10 specific DNA fragments from S. spontaneum, ZT51-439 was the highest with the rate of 95.92% and ZT61-678 was lowest at the rate of 2%. Fragment with the highest recovering probability of 25 specific DNA sequences from wild species of Saccharum spp. was ZT85-700 with the rate of 92.98%, while ZT82-460 was the lowest at the rate of 17.54%. 18 specific DNA fragments of Saccharum could transfer to progeny with the rate of 100%. 16 specific DNA sequences of Erianthus arundinaceus could transfer in F1 and F2 with the rate of 50%. The specific sequences ZT37-377, ZT40-928 of Saccharum genus and ZT52-439 of S.spontaneum were located on chromosomes of cultivar YG No.11 and Badila by using in situ PCR. The signals mainly distributed on the telomere, some on centromere and very few on whole arms. ZT37-377 was located on the 56 chromosomes of YG No.11 with No. of 1~12 and 14~32 respectively. ZT52-439 was located on only 29 chromosomes of YG No.11 with No. of 2, 5～7, 9～20, 23, 25 and 29 respectively. ZT40-928 was located on the 69 chromosomes of Badila with No. of 1~11, 13~19, 22~32 and 34~39 respectively.Karyotype on YG No.11 and Badila was analysed. The karyotype formula of YG No.11 was 2n=64=56m(2sat)+8sm. Its karyotype belonged to 2A type. The absolute length of chromosome was 1.54～2.95μm, while the relative length was 2.23%～4.28%. Chromosomes of No.19, 28, 30 and 32 were sub-median kinetochores, others were median kinetochores. The short arm of chromosome of No. 31 had one satellite. As to Badila, the karyotype formula was 2n=80=80m and belonged to 1B type. The absolute length of chromosome was 1.10～3.29μm, and the relative length was 1.39%～4.16%.The specific DNA sequences Aso64(378bp), Asp68(545bp)and Asp70(300bp), amplified by polymorphism primer ZT67, were compared by landing NCBI. The results showed the main differences between these specific DNA sequences of S. officinarum(Aso64)and S. spontaneum(Asp68,Asp70)were the deficiency or insertion of some fragments. The probable functions of 21 typical specific DNA sequences were also analyzed and it showed that 19 sequences were not congenetic to the sequence of known functions. Only 2 sequences were low congenetic to the sequences of known functions and their probable function was mostly relative to Sorghum bicolor clone SBTXS. Fingerprint of 96 sugarcane germplasm was constructed by ISSR with 7 primers and the cluster analysis of the 96 sugarcane germplasm was performed. The banding profiles of E. arundinaceus were obviously differentiated from that of Saccharum spp.. Among S. spontaneum, Yunnan spont., Yacheng spont. , Huanan spont. and Indian spont. were not clustered into one group. This indicated that variation was common and distinct in S. spontaneum.