Browsing by Author "Naidoo, Roobavathie."
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Item Development and application of SNP marker for low phytic acid gene (Ipa1-1) with studies on the effect of low phytic acid on seed germination, vigour and yield in maize.(2010) Naidoo, Roobavathie.; Tongoona, Pangirayi.; Watson, Gregory M. F.; Derera, John.; Laing, Mark Delmege.Maize grain contains high levels of phytic acid which chelates iron, zinc and other micronutrients as it passes through the digestive systems of monogastric consumers reducing their bioavailability. Breeding for low phytic acid (LPA) content to improve micronutrient bioavailability is hampered by a tedious and destructive colorimetric method on the grain, low yields compared to the wild-types and reduced seed germination and vigour of LPA mutants. Breeding for LPA therefore should also incorporate breeding for improved germination and vigour in the mutants. Molecular markers to speed up the selection process and studies on gene action and combining ability for germination, vigour and yield parameters of the LPA mutants in combinations with other different maize germplasm will speed up breeding for this trait. The objectives of this study were: to develop a molecular marker linked to the lpa1-1 gene and apply this marker for foreground selection in a backcross breeding programme and to use amplified fragment length polymorphism (AFLP) markers for background selection to recover the recurrent parent genome to speed up the backcrossing process; to study gene action and combining ability for seed germination, vigour and yield from diallel crosses involving LPA mutants, QPM and normal endosperm maize inbred lines by replicated laboratory seed tests ( standard germination test and accelerated aging test) and field evaluations in South Africa and Zimbabwe. A co-dominant single nucleotide polymorphism (SNP) marker which detects the transition base change of C/T nucleotides was developed from the gene sequence to identify the lpa1-1 trait. The 150 bp lpa1-1 SNP marker was validated by forward and reverse DNA sequencing of the parental amplification products which confirmed the C to T base change resulting in the LPA phenotype. The lpa1-1 SNP marker was used for foreground selection in 250 BC(2)F(1) progenies of CM 32 (LPA) x P 16 as the recurrent parent. This SNP marker was used to genotype the lines into homozygous dominant (wild type) and homozygous recessive (LPA) genotypes by their melting profiles and heterozygous genotypes by the normalised difference plots using high resolution melt (HRM) analysis. Seventeen heterozygous and 11 homozygous recessive lines were identified for background selection by fingerprinting with AFLP markers to determine the amount of recurrent parent (P 16) genome present. There were six EcoRI/MseI primer combinations tested with 277 data points scored (84% polymorphism rate). The amount of recurrent parent (P 16) genome recovered ranged from 62% to 92% with 13 lines showing greater than 83% of the recurrent parent genome. The effects of diallel crosses generated between four LPA, three QPM and three normal endosperm maize lines were determined for seed germination and vigour using the standard germination and accelerated aging seed tests under laboratory conditions in accordance with the procedures of the International Seed Testing Association. The specific combining ability (SCA) effects and general combining ability (GCA) effects were significant for the seed germination and vigour traits, indicating that genes with non-additive and additive effects were important in controlling these traits. However, the SCA effects were greater than GCA effects suggesting that genes with non-additive effects were predominant. The LPA parents showed reduced vigour compared to the normal and QPM inbred lines under both conditions, with LPA lines CM 31 and CM 32 showing stress tolerance. There were some combinations involving LPA lines, such as LPA x normal, LPA x QPM and LPA x LPA that retained high vigour and high germination rates under accelerated aging conditions, suggesting that they could be stress-tolerant.. A 10 x 10 diallel involving four LPA, three QPM and three Nm inbred lines was evaluated in replicated trials across six environments. Results show that both additive and non-additive gene effects were significant for resistance to northern corn leaf blight (NCLB), grey leaf spot (GLS) and Phaeosphaeria leaf spot (PLS) diseases. The additive gene effects were predominant for the yield and associated secondary traits such as days to mid-pollen shed (DMP), days to mid-silking (DMS), ear per plant (EPP) and grain moisture content (GMC) and grain yield. The LPA lines were early flowering and had quick grain dry down rate but all showed undesirable negative and significant GCA effects for yield. The yield of the LPA x LPA, LPA x Nm and LPA x QPM group of crosses was lower than the check hybrids by about 32 to 67% showing the need for yield improvement of the LPA combinations. An eight x eight diallel involving two LPA and six normal endosperm lines was evaluated over two seasons in five locations with two replications for grain yield components and foliar diseases. There was significant additive and non-additive gene action for both seed germination and vigour traits with predominance of non-additive gene effects. Both additive and non-additive gene effects were significant for yield and associated traits such as anthesis dates and number of ears per plant. However, the additive gene action was predominant for yield and associated traits. Generally the LPA lines and their combinations showed lower germination and vigour. The LPA line, CM 32 showed stress tolerance under accelerated aging conditions. There were three LPA x Nm crosses that showed improvements to the means of seed germination and vigour and yield traits. Results indicated that there was not any significant correlation between yield and seed germination and between yield and vigour. Yield was, however, significantly and positively correlated with anthesis dates and GMC, indicating that higher yield was associated with longer growing cycles. This study was able to successfully develop and apply the lpa1-1 SNP marker for foreground selection and AFLP markers for background selection in a backcross breeding programme. Problems of low seed germination, seedling vigour and grain yield of LPA lines and their combinations were confirmed. However results also indicated some potential of combining the LPA and QPM traits in a single cultivar. In general, procedures such as reciprocal recurrent selection, that emphasise both GCA and SCA effects would be recommended to improve seed germination, seedling vigour and yield in developing varieties with LPA trait.Item Development of a pepper (Capsicum annuum L.) hybrid variety with resistance to potato virus Y (PVY) using molecular breeding.(2013) Moodley, Vaneson.; Gubba, Augustine.; Naidoo, Roobavathie.Pepper (Capsicum annuum L.) is an important vegetable crop grown and consumed worldwide. Potato virus Y (PVY) is a globally economically important pathogen which significantly reduces the yield and quality of cultivated pepper. The virus is considered as a major limiting factor to the economic production of pepper in the province of KwaZulu-Natal (KZN) in the Republic of South Africa (RSA). Many applied practices to control the spread of PVY are ineffective to mitigate the losses incurred by many farming communities across the KZN province. Therefore, the objectives of this study was to determine the full genome sequence of a PVY isolate from KZN, to identify resistance alleles in commercially available pepper varieties in KZN and to develop a pepper hybrid variety with resistance to PVY using a molecular breeding strategy The first part of the study was conducted to determine the first full genome sequence of a PVY isolate (JVW-186) infecting pepper from KZN. The complete genome sequence of JVW-186 was assembled from overlapping RT-PCR clones using MEGA 5 software. Individual ORFs were identified using the nucleotide data base NCBI and aligned using CLUSTALW. RDP4 software was used to identify recombination junctions in the sequence alignment of JVW-186. CLC Main Workbench 6 software was used to determine the nucleotide sequence similarity of recombinant and non-recombinant fragments of JVW-186 in conjunction with ten PVY parental isolates. Based on sequence data, virus morphology and the coat protein size as determined by SDS-PAGE analysis, the identity of the isolate JVW-186 was confirmed as PVY. Phylogenetic trees were constructed from all recombinant and non-recombinant segments of the sequence by the maximum likelihood method using MEGA 5 software. The full length sequence of JVW-186 consisted of 9700bp. Two ORF’s were identified at position 186 and 2915 of the sequence alignment encoding the viral polyprotein and the frameshift translated protein P3N-PIPO, respectively. RDP4 software confirmed two recombination breakpoints at position 343 and 9308 of the sequence resulting in four segments of the genome. At each recombination event, a 1021-bp fragment at the 5’ end in the region of the P1/HC-Pro protein and a 392-bp fragment in the region of the coat protein shared a high sequence similarity of 91.8 % and 98.89 % to the potato borne PVYC parental isolate PRI-509 and the PVYO parental isolate SASA-110 respectively. The non-recombinant fragment 1 clustered within the C clade of PVY isolates; however the large 7942-bp fragment 3 did not cluster within any of the clades although it shared > 80% nucleotide sequence similarity to other PVY isolates used in this study. Our results suggest that isolate JVW-186 is a novel recombinant strain of PVY that could have evolved due to the dynamics of selection. The second part of the study aimed to evaluate different pepper lines for resistance to PVY. Two recessive alleles (pvr21 and pvr22) located on the pvr2-elF4E locus are known to confer resistance to the virus. To this end, six pepper lines were challenged with PVY infected Nicotiana tabacum cv. Xanthi leaf material using mechanical inoculation under greenhouse conditions. Each line was assessed for resistance to PVY by visual screening for disease severity and quantitative enzyme linked immunosorbent assay (ELISA) for virus load. Pepper lines were further characterized using tetra-primer ARMS-PCR (amplification refractory mutation system polymerase chain reaction) to identify and differentiate the presence of homozygous/heterozygous resistance alleles that confer PVY resistance. Evaluations revealed two resistant pepper lines (Double Up and Cecelia) and varying levels of susceptibility in the other four pepper lines challenged with PVY. The most susceptible pepper line was Benno, although high levels of susceptibility were observed in three other lines (IP, Mantenga and Excellence). The pvr2+ allele was positively identified in all the susceptible pepper lines using the T200A tetra-primer which confirms that the presence of this allele is dominant for PVY susceptibility. Double Up and Cecelia were genotyped homozygous pvr21/pvr21 and pvr22/pvr22 respectively, and remained asymptomatic throughout the trial which indicates that these alleles confer resistance to the isolate of PVY used in this study. The information generated in this study can be incorporated into breeding programs intended to control PVY on pepper in KZN. The final part of the study focused on the development of resistant varieties as the best alternative to manage PVY diseases on pepper. Homozygous F2 pepper lines were developed from local germplasm carrying PVY resistance genes (pvr21 and pvr22) using marker assisted selection (MAS). The F1 progeny was obtained by crossing a homozygous pvr21 (resistant) ‘Double Up’ cultivar with a heterozygous susceptible (pvr2+/pvr22) ‘Benno’ cultivar. F1 and F2 generations were assessed for the presence of PVY resistance/susceptibility alleles (pvr2+/pvr21/pvr22) at the pvr2-elF4e locus using the tetra primer amplification refractory mutation system – polymerase chain reaction (ARMS-PCR) procedure. Negative selection was carried out using the tetra-primer T200A marker to detect the pvr2+ (susceptible) allele. All F1 progeny displaying the pvr2+ allele were eliminated from further study. All 302 plants belonging to 29 F2 families expressing homozygous recessive traits were tested via mechanical inoculation for their response to PVY infection and resistance to PVY was confirmed in all selected families based on symptomatology in greenhouse house screens using double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). These results show that ARMS-PCR can be used to successfully screen pepper genotypes for alleles that confer PVY resistance thereby contributing to the improvement of pepper production using molecular breeding approaches.Item Marker-assisted selection for maize streak virus resistance and concomitant conventional selection for Downy Mildew resistance in a maize population.(2013) Mafu, Nothando Fowiza.; Laing, Mark Delmege.; Derera, John.; Naidoo, Roobavathie.Maize streak virus (MSV) disease, transmitted by leafhoppers (Cicadulina mbila, Naude), and maize downy mildew (DM) disease caused by Peronosclerospora sorghi (Weston and Uppal) Shaw, are major contributing factors to low maize yields in Africa. These two diseases threaten maize production in Mozambique, thus the importance of breeding Mozambican maize varieties that carry resistance to these diseases. Marker-assisted selection (MAS) was employed to pyramid MSV and DM disease resistant genes into a single genetic background through simultaneous selection. Firstly, it was essential to determine the genetic diversity of MSV disease resistance in 25 elite maize inbred lines to aid in the selection of suitable lines for the introgression of the msv1 gene; and subsequently, to introduce the msv1 resistance gene cluster from two inbred lines, CM505 and CML509, which were identified as the ideal parental lines for the introgression of MSV disease resistance into a locally adapted Mozambican inbred line LP23 that had DM background resistance. Pyramiding the resistance genes by the use of simple sequence repeat (SSR) molecular markers to track the MSV gene cluster was investigated in 118 F3 progeny derived from crosses of CML505 x LP23 and CML509 x LP23. High resolution melt (HRM) analysis using the markers umc2228 and bnlg1811 detected 29 MSV resistant lines. At the International Maize and Wheat Improvement Centre (CIMMYT) in Zimbabwe, MSV disease expression of the 118 F3 progeny lines was assessed under artificial inoculation conditions with viruliferous leafhoppers and the effect of the MSV disease on plant height was measured. Thirty-seven family lines exhibited MSV and DM (DM incidence ≤50) disease resistance. Individual plants from a total of 41 progeny lines, that exhibited MSV disease severity ratings of 2.5 or less in both locations within each of the F3 family lines, were selected based on the presence of the msv1 gene based on SSR data, or field DM disease resistance, and were then advanced to the F4 generation to be fixed for use to improve maize hybrids in Mozambique for MSV resistance. Simultaneous trials were run at Chokwe Research Station in Mozambique for MSV and DM disease assessment, under natural and artificial disease infestation, respectively. Thus the MSV and DM genes were effectively pyramided. Lines with both MSV and DM resistance were advanced to the F4 generation and will be fixed for use to improve maize hybrids in Mozambique for MSV and DM resistance, which will have positive implications on food security in Mozambique. This research discusses the results of combined selection with both artificial inoculation and the three selected SSR markers. It was concluded that a conventional maize breeder can successfully use molecular markers to improve selection intensity and maximise genetic gain.