The Physical Locations Of Neutral Or Alkaline Invertaes Gene Family In Manihot Esculenta Crantz

Cassava (Manihot esculenta Crantz) originating in South America is an important food crop and energy plant in the tropics. Neutral or alkaline invertase is a key enzyme in the process of cassava starch accumulation. 11 members of neutral or alkaline invertase genes family (MeNINV1?10 and nINVl) from cassava have been cloned by predecessors,and their structure evolution and expression also have been analyzed. But there are few reports about the physical localization on chromosomes of them.In this study, chromosome specimens were prepared from the bronze-coloured tender leaves of cassava (SC6), and the 10 members of the cassava neutral or alkaline invertase gene family were physically positioned by fluorescence in situ hybridization and in situ PCR.The distribution characteristics and the linkage with other genes are clarified. The results are as follows:(1) The method of preparing chromosome specimens from the bronze-coloured tender leaves of cassava was optimized. It was found that the optimum pretreatment method was treated with saturated p-dichlorobenzene solution for 1.5 h. The optimum enzymatic solution was enzymolyzed in a mixture of 5% cellulase and 4% pectinase at 37 ? for 4.5 h.(2) The rinsing steps after hybridization were optimized. The results show that when the time of three rinsing is controlled to 5 min, 5 min and 8 min, the background of the field of view is the most clean.(3) The members of the cassava Neutral or alkaline Invertase Gene family were physically positioned by single-color fluorescence in situ hybridization, two-color fluorescence in situ hybridization and in situ PCR. The results shows that MeNINV5,MeNINV9, MeNINV10 genes are located on chromosome 4. MeNINV9's and MeNINV10's percentage distances of the signal locus to the centromere are 68.52 and 95.35 on the short arm respectively. MeNINV5's percentage distances of the signal locus to the centromere is 22.71 on the long arm. MeNINV4 and nINV1 genes are located on the long arm of chromosome 6, the percentage of the centromere distance are 43.16 and 80.71 respectively.MeNINV6 and MeNINV7 genes are located on the long arm of cassava(SC6) chromosome 7 and 17 respectively, and the percentage distances of the signal locus to the centromere are15.38 and 57.97 respectively; MeNINV2, MeNINV3 and MeNINV8 genes are located on the short arm of cassava(SC6) chromosome 9, 5 and 16 respectively, and the percentage distances of the signal locus to the centromere are 51.88, 91.75 and 76.33.(4) The relationship between the members of the neutral or alkaline invertase gene family was analyzed: MeNINV5, MeNINV9 and MeNINV10 genes are all located on cassava(SC6) chromosome 4, they are linked genes; MeNINV4 and nINV1 genes are located on the long arm of chromosome 6, they are linked genes to each other as well; however, other genes are located on different chromosomes respectively. They are inherited independently.(5) The relationship between the members of the neutral or alkaline invertase gene family and the genes that have been previously identified was clarified in this study: The results showed that MeNINV6, SBE? and SUT4.1 genes are located on chromosome 7,MeNINV1 and GBSSII genes are located on chromosome 11; MeNINV2 and SBEI genes are located on chromosome 12; MeNINV7 and SUT2 genes are located on the Chromosome 17.