Study On ISSR Marker Of Mango And Its Application In Early Identification Of Elitemango Plant

Mango (Mangifera indica L) is one of the five main tropical fruits in the world ,which is also famous tropical fruit in our country. Because genetic relationships among our main mango cultivars and native mango cultivars in our country were unclear, and early screening methods for elite mango plants were also lacked, it is very necessary to obtain some methods for assessing genetic relationship of mango and early identification of good cultivars. In this study, ISSR marker which is a more powerful, rapid, simple and inexpensive molecular method was used in assessing genetic relationship and diversity, distinguishing closely related cultivars of mango, identifying physiological disease and anthracnose of mango. The main results were as follows:1. The high quality genomic DNA was obtained with our improved CTAB method, chloroplast DNA (cp DNA) was extracted from purified chloroplast that isolated chloroplast was pured by saccharose grades of 70%.60%. 45%.30% with our improved CTAB method2. The optimal ISSR reaction system and amplification procedure for mango were founded. The optimal ISSR reaction system was each 20μL, amplification reaction consisted of 10mM Tris-HCl (pH 8.3), 50mM KCl, 1.5mM MgCl₂, 0.2mM mixed dNTP, 0.25μM primer, 1 unit rTaq polymerase (Takara Biotechnology, Japan ), and approximately 60ng genomic DNA or chloroplastic DNA. The optimal PCR amplification procedure in Thermcycler was as follow: 5min at 94℃for 1 cycle, followed by 1min at 94℃, 1min at 47℃-53℃(50℃-55℃for cp DNA) and 2min at 72℃for 40 cycles, and 10min at 72℃for final extension.3. Twenty ISSR primers screened from 120 primers, with which ISSR-PCR could produce reproducible, polymorphic DNA amplification patterns, were selected for genomic DNA. Eight primers of 50 primers were good for cpDNA ISSR-PCR. among the 8 cpDNA ISSR primers, the 7 cpDNA ISSR primers are the same as the primers for genomic DNA ISSR-PCR.4. Seven carabao mango cultivars or lines and Liuzhou lusong mango were examined with ISSR primers. Of the 30 primers screened, 6 primers gave reproducible, polymorphic DNA amplification patterns, and were selected to construct a DNA fingerprinting map to distinguished carabao mango cultivars or lines. According to the banding patterns, all cultivars tested in this study were distinguished from each other by every one of 6 selected primers and showed ample genetic diversity. Based on UPGMA analysis the carabao showed the lowest similarity to all other cultivars while Gaozhou carabao, Zhanjiang carabao, Tianyang xiangmang, Jinqianmang, Liuzhou lusong, Yueximang No.1and Panxi red carabao could be clustered into one group.indicating thatISSR-PCR was an effective method for cultivar identification of mango cultivars and lines.5. Thirty-eight mango cultivars, Mangifera himalis J.Y. Liang and Mangifera persiciformis Wu & Ming, including 23 Guangxi native, 4 India, 3 USA, 3 Burma, 2 Thailand , 1 Philippines, 1 Pakistan and 1 Sir Lanka cultivars, were examined with ISSR primers. Of the 42 primers screened, 9 primers were selected to construct a DNA fingerprinting map to distinguished the genotypes of mango. Based on 74 selected bands, all Guangxi mango cultivars tested were clustered into a big group with Neelum, Macheso, Aroemwnis, and Irwin by UPGMA analysis, indicating that Guangxi mango cultivars had a close relationship each other.6. Thirty-three mango cultivars and two relative species M. Himalis J.Y. Ling and M. Persiciformais C.Y. Wu et T.L. Ming collected from Guangxi Academy of Agricultural Sciences were examined by ISSR-PCR with genomic DNA and cpDNA. Compared ISSR electrophoresis patterns amplified with 7 common primers, The cpDNA ISSR-PCR obtained more polymorphic bands than genomic DNA ISSR-PCR and the average percent of polymorphism was 79.4% vs. 70.7%. If coefficient 0.73 of UPGMA analysis with 7 common primers was regarded as divided line of genetic relationship of mangoes, 35 samples could be clustered into four groups by cpDNA ISSR or two groups that were M. indica group and M. persiciformis group by genomic DNA ISSR. In extent that The result showed that was more reasonable for assessing genetic relationship of mangoes by cpISSR than by genomic ISSR.7. The nine ISSR primers of 20 ISSR primers were used in PCR amplification to distinguish rapidly fruit physiological disease of mango in molecular level. The study indicated that 20 mango cultivars were divided into two groups , which 8 mango cultivars infected easily by physiological diseases were clustered into on group, and the others were composed of another group. It was said that the ISSR technique could be used as a method of early identification of physiological disease in mango.8. The resistance of twenty mango cultivars to anthracnose was studied at the molecular level by Inter-simple Sequence Repeat (ISSR). Of the more than 120 ISSR primers screened, 8 ISSR primers were selected as PCR amplification and cluster analysis. The result showed that the twenty mango cultivars were clustered into three groups which were markedly correlated with the field resistance of mango cultivars to anthracnose, indicating that the ISSR markers can be used as a useful method for early screening the anthracnose resistance cultivar in mango breeding.