IDENTIFICATION OF NEW MOLECULAR MARKERS FOR DIVERSITY ANALYSIS AND BREEDING FOR EARLY MATURITY AND DETERMINATE LABLAB (LABLAB PURPUREUS) VARIETIES

Abstract

Lablab (Lablab purpureus (L.) Sweet) is a grain legume crop commonly grown in Africa and India and is used as human food, animal feed, in soil conservation, enhancing soil fertility and in weed management. In Kenya, most farmers grow landraces which are inherently low yielding and have other undesirable attributes like long maturity duration and indeterminate growth habit. The status of genetic diversity of the local lablab germplasm and how it relates to materials from other regions remain unclear. Unavailability of novel breeding selection tools such as molecular markers and lack of adequate information on the inheritance pattern of important traits have also hampered the crop improvement in Kenya. The objectives of the study were therefore: to develop new molecular markers for lablab; to assess the genotypic diversity of local, exotic and wild lablab accessions using simple sequence repeats, diversity array technology (DArT) and single nucleotide polymorphism (SNP) molecular markers; to identify the inheritance pattern of selected lablab qualitative traits and establish the linkage relationship of the genes controlling them; determine the heritability estimates, genetic gain and character association of important traits of determinate lablab. Transcriptome sequencing using 454 Titanium FLX system of mRNA isolated from leaves and shoots of lablab samples, was conducted to discover genic-SSRs and to develop SSR markers. Eight of these new developed SSR markers were used to characterize 189 lablab accessions. SilicoDArT and SNP markers were developed using DArTSeq technology and used to characterize 240 lablab accessions. The genetics of growth habit and other qualitative traits were studied in three generations (F1, F2 F3) of eight lablab populations. Selected F5 lines with determinate growth habit were grown using RCBD design at KALRO Thika and Katumani to determine heritability estimates, genetic gain and character association. Results indicated that there were 446 genic SSRs from 3140 assembled lablab contigs indicating an overall density of 202 SSR per Mbp. SSR primer pairs designed from the contigs sequences amplified on lablab genome. The gene diversity among the 189 accessions based on SSR loci ranged from 0.26 to 0.52 with an average of 0.38, with germplasm collected from Kenya showing a moderate genetic diversity of 0.36. Higher genetic diversity (He<0.5) was detected within the Ethiopian and South Africa populations. A total of 15,601 polymorphic DArT markers and 11,431 SNP markers were identified each with average reproducibility and genotype call rate of more than 90%. Based on both DArT and SNP markers the 240 lablab was of narrow genetic diversity with the expected mean heterozygosity of 0.030 (DArT) and 0.039 (SNP). However, genetic differentiation was most pronounced between the cultivated and the wild accessions. The growth habit in lablab is under control of three genes which could be temperature dependent. The genes controlling stem growth habit and time to flowering in lablab are linked. The study identified, moderate to high heritability, genetic advance estimates and significant positive correlations of pods per plant, raceme per plant, plant height, pod width, racemes per pod and number of flower nodes. The newly developed molecular markers are useful in grouping lablab genotypes into related clusters that breeders can use to enhance lablab productivity. Selection for high number of units of pods per plant, raceme per plant, plant height, pod width, racemes per pod and can be effective when targeting to develop high seed yielding determinate varieties.