The objective of the present study was to investigate the potential use of bulk segregant analysis (Michelmore et al., 1991) as a method to rapidly identify genetic markers linked to traits in sugarcane. Four bulked DNA samples were prepared from progeny of a sugarcane cross, AA157, based on segregation for the fibre trait. The bulks comprised five and ten individuals on either side of the fibre phenotypic extreme. The random amplified polymorphic DNA (RAPD) technique (Williams et ai., 1990) was used to screen for differences between the low and high fibre bulks. A total of 749 fragments were amplified in the bulks, eight of which were polymorphic. The segregation of the bulk specific polymorphism was analysed in 80 progeny of the AA157 cross; and seven were found to reproducibly segregate on a 1: 1 basis. This indicates that they are single dose fragments. A total of 79 polymorphisms were detected between the parents of the cross, indicating 10.5% variation in the genomic region sampled. Twenty two of the parental polymorphisms segregated as single dose fragments in the progeny of the cross AA157. Analyses of variance (ANOVAs), and multiple regression analyses, were used to ascertain linkage of the putative RAPD markers to fibre, and if linked, to determine the fibre variation ascribed respectively. Three RAPD fragments were found linked to the fibre trait. Fragments OPA17438 and OPC16889 (at the 99% significance level), and OPB1l464 (at the 95% significance level). These putative markers ascribed a total of 28.6% fibre variation in the 1993 season. The association of the RAPD markers with fibre in the different seasons (1992, 1993 and 1994) was investigated. Three RAPD markers were found linked to the fibre trait in each season, with a total of 5.5% and 31,4% fibre variation ascribed in the 1992 and 1994 seasons respectively. Marker OPA17438 was found to be linked to the fibre trait in all three seasons investigated, and marker OPC16889, was found linked to the fibre trait in the 1992 and 1993 seasons. Cross validation of the linkages of the RAPD markers to the fibre trait was carried out by a modified form of 'jacknifing' where the sample size was reduced to N-l0, and RAPD marker-fibre trait associations investigated as before. RAPD markers OPA17438 and OPC16889 were still consistent across the seasons, however marker OPA17438 was no longer linked to the fibre trait in the 1992 season. To investigate the genetic behaviour of RAPD based markers in sugarcane and the potential for their application in marker-assisted selection (MAS), two putative RAPD markers were converted to sequence characterised amplified regions (SCARs) (Paran and Michelmore, 1993). The RAPD fragments OPA17438, OPBl1464, and OPC16889 were excised from agarose gels, re-amplified and cloned into the pCR-Script SK (+) phagemid for sequencing. RAPD markers OPA17438 and OPB11 464 were converted to SCARs by using their sequences to design longer specific primers. A third SCAR marker, SAl1640, originally derived from sugarcane cDNA as a potential stem preferential expressed sequence tag, was included in the analysis to increase the sample size. All three SCAR markers segregated in a monomorphic fashion in the parents and progeny of the cross AA157. In addition, monomorphic length variants for markers, OPA17438 and OPB11 464 were detected with the SCAR amplification. All three SCARs segregated in a monomorphic fashion in different commercial varieties and bulks of S. officinarum and S. spontaneum, the progenitors of modern commercial varieties. The segregation analyses of the SCAR markers indicate that the RAPD polymorphism of marker SAl1640 was probably due to a point mutation or mismatch in the priming site. The segregation analyses of SCARs for the markers OPA17438 and OPB11464 indicate that their segregation in the RAPD analyses was due to an insertion mutation in the genetic locus. The combined results of the SCAR and RAPD segregation of markers OPA17438 and OPB11464 are indicative of preferential pairing in the cross AA157. Finally, to investigate the extent of linkage disequilibrium in a modern commercial variety, twenty two single dose RAPD fragments were investigated for their association with four traits in 53 progeny of cross AA157. The four traits investigated were fibre %cane, brix %cane, pol %cane and ers %cane over three seasons (1992, 1993 and 1994), at different ages of harvest (12, 8, and 9 months respectively). Seventeen linkages of RAPD markers to the four traits, over the three seasons, were detected. The phenotypic variation ascribed by the RAPD markers ranged from 7.6% fibre %cane variation explained by one marker in 1992, 29.6% fibre %cane (three markers) in the 1993 season to 10% (three markers) in 1994. A total of 14.1% brix %cane variation was ascribed by two markers in 1992, 9.6% (one marker) in 1993 and 16.3% (two markers) in the 1994 season. A total of 13.5% estimated recoverable sucrose %cane was ascribed by one marker in 1992, 12% (two markers) in 1993 and 15.3% (two markers) in the 1994 season. Two markers explained 17.2% pol %cane variation in 1992 and 25.4% in the 1994 season. Only four markers were detected across different environments, three of which were linked to fibre. These were OPA17438, OPB16618 and OPC16889, each linked to fibre in two seasons. RAPD marker OPB11 464 was linked to estimated recoverable sucrose %cane in two seasons. Two markers were found associated with different traits in a single season. RAPD marker OPB11 464 was found associated with brix %cane and estimated recoverable sucrose %cane in the 1993 season, and RAPD marker OPA17438 was found associated with all four traits in the 1994 season. / Thesis (Ph.D.)-University of Natal, Durban, 1998.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005. / Vacuolar Pyrophosphatase (V-PPase) has never been studied in sugarcane before and to date nothing is known about V-PPase in sugarcane, except for the sequences of a few expressed sequence tags (ESTs). The aim of this project was to characterize V-PPase expression in several hybrid sugarcane varieties that differ significantly in sucrose content, with the main objective of the study to assess whether V-PPase is correlated in any way to the sucrose storage phenotype. Therefore, the goals of this project were to (i) develop molecular tools for the detection and quantification of V-PPase on a DNA, RNA, protein and enzyme level and (ii) to use these tools to characterize the expression of V-PPase within the culm of the three hybrid varieties. The cDNA sequence of the catalytic subunit of the sugarcane V-PPase gene was cloned, expressed in a bacterial system and the V-PPase peptide was purified. This peptide was used for the immunization of mice and the production of polyclonal anti-VPPase antiserum. Anti-VPPase antiserum reacted specifically with a single polypeptide among vacuolar membrane proteins. Moreover, anti-VPPase antiserum recognized V-PPase from various monocotyledons and dicotyledons. The anti-VPPase antiserum was used for the establishment of an ELISA system to determine V-PPase protein content in vacuolar membrane preparations. This system proved to have several advantages over the protein blotting technique and shared a strong linear relation with V-PPase specific activity, showing that these two tests are compatible and reliable. The optimisation of sugarcane V-PPase zero-order kinetics was fundamental in order to measure V-PPase specific activity accurately. It had a relative broad pH optimum, retaining more than 90% of its maximum activity between pH 6.50 and 7.25. V-PPase required both Mg2+ and K+, in addition to PPi, for maximum activity in vitro. The reported kinetic variables are within range of previous data determined for other species, including mung bean, red beet and sugar beet. V-PPase protein level and specific activity within the sugarcane culm followed a similar trend , withoiofofoenaobserved for sucrose accumulation rates observed in sugarcane. Moreover, V-PPase protein contents and specific activity share the same general trend as total sucrose content in a specific tissue compared among the three varieties. No significant differences were observed in V-ATPase activity among the three varieties. Our findings suggest that V-PPase may play a role in sucrose accumulation in sugarcane.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) is an important glycolytic enzyme and catalyses the reversible conversion of fructose-6-phosphate (Fr-6-P) and pyrophosphate (PPi) to fructose 1,6-bisphosphate (Fr-1,6-P2) and inorganic phosphate (Pi). Sugarcane PFP has been inversely correlated with sucrose content across segregating F1 varieties. The down-regulation of PFP in cultivar NCo310 in a previous study led to an increase in sucrose accumulation and fibre content in immature tissue. Several potential transgenic sugarcane lines from genotypes 88H0019 and N27, transformed with the untranslatable sense sugarcane PFP-β gene, were characterized in this study. Initial screening for transgenesis was determined by slot blot and Southern blot analysis to confirm the presence of the co-transformed selectable marker npt II transgene. Northern blot analysis confirmed expression of the 1.2 kb PFP-β transcript in 7 of 9 lines analyzed. Sugar analysis using standard South African Sugarcane Research Institute (SASRI) mill room practices and HPLC was performed on 12 month old pot grown stalks divided into immature and mature tissue sections. The analysis of wild type 88H0019 showed an average sucrose content of 17.84 and 30.76 g sucrose/stalk in immature and mature tissue, respectively. However, no significant difference between the putative transgenic plant values and wild type controls was seen. PFP specific activity was determined in these tissues using enzymatic assay analysis and although levels obtained in immature tissue were between 5-18 nmol/min/mg protein, they were less than values previously reported in sugarcane. The results indicated that no down-regulation of PFP in immature tissue occurred when comparing transgenic and wild type plants. A more discrete internodal tissue sampling method was used to overcome the difficulty of detecting small changes in PFP enzyme activity in bulked stalk tissue sections. Fine analysis of PFP was conducted on specific developmental tissues and single stalks were divided into immature (internodes 1-3), maturing (internodes 4-5) and mature (internodes 7-8) regions. Sucrose analysis was performed using HPLC and PFP activity was determined enzymatically on each tissue type. The analysis of discrete developmental tissues showed specific PFP activity of 60-80 nmol/min/mg protein in young tissue, an amount which falls in the range previously obtained for sugarcane. However there was no significant difference between PFP or sucrose in the transgenic lines when compared with the wild type controls in any of the three developmental tissues examined. Western blotting and densitometric analysis of the blots confirmed the lack of PFP down-regulation in immature tissue in all lines. A final analysis of PFP iv in immature stalk tissue on selected lines was performed using quantitative PCR, which became available near the end of the study. The fold change of each transgenic line indicated that there was a minor increase in PFP confirming the lack of effect of transgenesis. Although evidence for the expression of the PFP-β transgene was seen in the northern blot, no further evidence for transgenesis could be found to support the desired effect of down-regulation of PFP. Characterization of transgenic stalks in this study was hindered by a limited number of lines available for analysis and large variability between replicate samples. Sampling techniques employed in an attempt to make use of existing standard SASRI mill room practices for sugar analysis highlighted the need for a more precise sampling method, specifically when determining the effects of an enzyme manipulation such as PFP. A refined approach has been developed which will assist researchers in the choice of analytical techniques for screening and characterization of potential transgenic lines in the future.

Thesis (MSc (Genetics. Plant Biotechnology)) --Stellenbosch University, 2008. / The main aim of the work presented in this thesis was to further our understanding of the role of Pyrrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) in sugarcane, by specifically investigating its potential contribution to phloem metabolism. PFP activity in sugarcane internodal tissue is inversely correlated to sucrose content across varieties that differ in their sucrose accumulation abilities. This apparent correlation is in contrast to previous studies that suggest PFP plays an insignificant role in metabolism. In the first part of this study an immunological characterisation of the two subunits of sugarcane PFP was conducted to establish whether it differ significantly from other plant species in terms of size and distribution. Both the alpha and beta subunit appears to be approximately sixty kilo Daltons in size and uniform in their relative distribution to each other in the various plant organs of sugarcane. Although the observed alpha subunit size is less than that predicted this could be explained at the hand of post translational modification, in essence the sugarcane PFP subunits appear similar than that described for other plants especially that of tobacco which was employed as a model system later on in this study. The only direct way to investigate PFP’s contribution to phloem metabolism is to alter its activity by recombinant DNA technologies. Therefore, in the second part of the study transformation systems were designed for both the constitutive and phloem specific downand up-regulation of PFP activity. For the down-regulation of activity a post transcriptional gene silencing system, i.e. a complementary strand intron hairpin RNA (ihpRNA) silencing system, was employed. A partial sequence of the PFP-beta subunit was isolated and used in vector construction. For the over-expression the Giardia lamblia PFP gene was used. The model plant tobacco was employed to investigate PFP’s effect on phloem metabolism and transport of assimilate. Transgene insertion was accomplished by means of Agobacterium mediated transformation and tissue specific manipulation of PFP activity was confirmed by in situ activity staining.