BREEDING MAIZE (Zea mays L.) FOR RESISTANCE TO AFLATOXIN ACCUMULATION IN GHANA
Maize (Zea mays L.) is the most important cereal crop in sub-Saharan Africa which provides food for more than 1.2 billion inhabitants. It is produced on nearly 100 million hectares in developing countries. However, production is hampered by many factors including low yields and aflatoxin contamination. Aflatoxins are carcinogenic secondary metabolite produced mainly by two species of Aspergillus (A. flavus and A. parasiticus). The toxin contaminates maize during pre-harvest as well as during storage. Grains with contamination levels above 20 ppb are destroyed in the USA while EU and Japan allows 2 - 4 and 0 ppb respectively. Ghana lacks regulatory infrastructure for monitoring and detecting aflatoxin in grains prior to market and moreover, most of the local maize varieties are susceptible to aflatoxin accumulation coupled with no practical mitigating activities to reduce aflatoxin accumulation in grains by consumers. Based on these challenges, this study was designed to (1) assess the levels of aflatoxins in maize and the extent of A. flavus distribution and diversity across 44 towns within 3 agro-ecologies where maize is actively cultivated, (2) assess diversity and screen both known aflatoxin resistant and local inbred lines (via phenotyping) for resistance to aflatoxin accumulation using local strain of A. flavus, (3) identify hybrid crosses that combine high yields and resistance to aflatoxin accumulation from crosses between locally adapted inbred parents and exotic resistant lines,(4) estimate GCA, SCA of parents and hybrid stability as well as heritability of traits and (5) introgress aflatoxin resistant QTLs from resistant donors into local inbred lines using marker assisted backcrossing.
Maize and soil samples collected separately from 34 and 44 towns respectively, were assessed for aflatoxin levels using HPLC technique. Distribution of A. flavus was determined using Misra and Miles approach while diversity was assessed using Darwin version 6.0.15 package. Seventeen local (susceptible) and 19 exotic aflatoxin resistant lines were evaluated for resistance
to aflatoxin accumulation, agronomic characteristics and genetic and morphological diversity. Thirty-six thousand and twenty SNPs were used to define the population structure and assign inbreds into respective heterotic groups. One hundred and sixty hybrids were subsequently generated from 10 local and 16 aflatoxin resistant inbreds using NCII design and evaluated across six environments for yield, aflatoxin accumulation resistance and stability. Furthermore, 20 polymorphic SSR markers were utilized in a marker assisted backcrossing approach to monitor foreground markers in successive generations to the BC2F1 stage where alleles linked to the aflatoxin accumulation resistant QTLs would be in the heterozygous phase within the background of 3 local inbreds were determined and successfully selfed.
Aflatoxin contamination in stored maize grains was wide spread across the ecological zones and some communities displayed dangerously high levels far beyond permissible limits set by Japan, European Union and the United State of America. Furthermore, significant variations were found in the strains of A.flavus isolates as well as their distribution across 44 communities sampled. Inbred lines MP715, MP719 and MP705 displayed significant and consistently low aflatoxin accumulation levels. Both morphological and genetic diversity between and among the genotypes were significant. Two and seven main clusters were observed for both morphological and genetic groupings at genetic similarity levels of 0.2 and 0.52 respectively. Furthermore, high corroboration was observed between clustering of inbreds based on STRUCTURE software version 2.3.4 and Power Marker version 3.25. Majority of inbreds clustered based on origin. Consequently, all data collected from the evaluation of 160 hybrids were analyzed using PROC GLM procedure of SAS software, version 9.3 (SAS Institute, 2008).
The statistical analysis showed significant effect of environment and genotypes for all traits especially aflatoxin accumulation and yield. The general combining ability of males for all traits were significant (P < 0.05) whereas that of the females were not significant for all traits. Inbreds with consistent significant negative GCA effect for aflatoxin reduction were identified (MP715, TZI8, MP719) and hybrids which combined high yields with reduced aflatoxin levels below 20ppb were developed. The superior stable hybrids identified out yielded the best check by 18% 13% and 15.2% respectively. Furthermore, the underlying genetic control for aflatoxin accumulation resistance trait was contributed by both GCA and SCA effects. Heritability estimates were fairly moderate suggesting permissible transfer of traits during selection. This means that it is possible to produce high yielding aflatoxin resistant hybrids for consumers.
Results from the marker assisted backcrossing led to the identification of a set of 20 polymorphic SSR markers which are tightly linked to important aflatoxin accumulation resistant QTLs which could effectively be utilized for foreground QTL introgression through marker assisted backcrossing. A total of 151 out of 500 BC1F1 families of five populations genotyped for foreground markers incorporated at least one target QTL into the genome. One large QTL was successfully incorporated into 6 BC2F1 families of ENT 70 x MP313E while a total of 2 QTLs from the MP715 and MP719 source were incorporated into 6 BC2F1 progenies. Two other progenies from the MP719 source incorporated one major QTL into the genome. Successful introgressions will serve as key candidates necessary for aflatoxin accumulation resistance breeding in Ghana.