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.
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.