| dc.description.abstract | Sugarcane smut 111 Kenya cause severe losses in susceptible varieties. Seedling smut
screening may select for sugarcane genotypes with physiological resistance, and may
therefore be of great use to the industry because genotypes with physiological smut
resistance are sought for commercialization. This study was conducted in the green house
first to assess the feasibility of inoculating sugarcane (Saccharum spp) seedlings with
smut (Usti/ago scitaminea syd), to evaluate different inoculation methods and secondly to
screen DNA markers for smut resistance. Crosses involving two populations emanating
from resistant and susceptible varieties was undertaken at Sugarcane Breeding Center-
Mtwapa(Mombasa).To investigate the reaction of seedlings to smut, three different
inoculation methods were employed. The first method involved soaking seedlings in smut
spore suspension at a concentration of 4 x 106 spores/ml for 30 minutes. The second
method involved wounding the seedlings at the bud with a scalpel then applying a paste
of smut made at a concentration of 2 grammes of spore for 2 ml of sterile water. The third
method was a paste method that involved a paste of smut at the seedling buds. Each
treatment had 30 entries (seedlings) planted in plastic bags in the glass house. Two
controls of the un-inoculated progenies were included. The experiment was randomized
complete block design replicated three times. There was no significant difference in whip
production between the two populations. Population Co 331 X Co 945 had near
significant results with wound paste method leading on whip production followed closely
by paste method. Inoculation had significant effect on seedlings survival across four
months under observation. There was significant difference in tiller production. There
was significant difference in whip production between the two families at three months.
The study recommends screening for smut resistance at first stage of selection to assess
seedlings reaction to smut and to avoid carrying large numbers of clones that are
eventually discarded at the advanced stage of selection. To screen molecular markers for
smut resistance, tissues of the seedlings were harvested and DNA extracted, quantified
and electrophoresis performed. DNA extraction method using lyophilized leaves was
better than sap extraction method in terms of DNA quantity and quality. Sugarcane tissue
gave a lot of DNA. The primers used in the experiment were obtained from SUCEST
database with expected homology of protein like Kinase. Another primer XLRR was
designed from conserved motifs of LRR, NBS and kinase domain. The pnrners were
synthesized by the oligo sequencing unit at the International Livestock Research Institute
(lLRI). The concentration of primers used in this study was between 200-300ng. DNA
templates of 30ng gave fairly good PCR products with clear bands. Only one hot- start
PCR temperature profile with a ramped decrease in annealing temperature was necessary
to display amplicons of high quality for the primer pairs tested. The amplicons in the
figures presented in this study represent the presence of segment that contains resistance
gene analog. Empty wells without any band indicate there was no amplification and
therefore the gene analog was absent. The bands were scored as present or absent
depending on their intensity and clarity. DNA extracted from plants that exhibited smut
whips phenotypically did not produce any band when PCR was done. Every primer pair
produced scorable loci in the progeny analyzed. The size of the product from primer SCB
07 and SCC09 was around 800bp. However the size of amplification products from
primer XLRR was near the expected size 900bp. Primer SCC09 produced very clear
bands. | en |
