Applying molecular genetics to underutilised species – problems and opportunities

Molecular markers represent an important tool for marker-assisted breeding in major crop plant breeding programmes. Applying molecular genetics to underutilised and minor crop species is more challenging as the funds available to research and develop such crops are often severely limited. Bambara groundnut is an underutilised African legume crop with good drought tolerance. It is also grown at low levels in Southeast Asia. In this review we examine some of the applications of DNA markers and illustrate their value in bambara groundnut

Increasing leaf vein density by mutagenesis: laying the foundations for C4 rice

A high leaf vein density is both an essential feature of C4 photosynthesis and a foundation trait to C4 evolution, ensuring the optimal proportion and proximity of mesophyll and bundle sheath cells for permitting the rapid exchange of photosynthates. Two rice mutant populations, a deletion mutant library with a cv. IR64 background (12,470 lines) and a T-DNA insertion mutant library with a cv. Tainung 67 background (10,830 lines), were screened for increases in vein density. A high throughput method with handheld microscopes was developed and its accuracy was supported by more rigorous microscopy analysis. Eight lines with significantly increased leaf vein densities were identified to be used as genetic stock for the global C4 Rice Consortium. The candidate population was shown to include both shared and independent mutations and so more than one gene controlled the high vein density phenotype. The high vein density trait was found to be linked to a narrow leaf width trait but the linkage was incomplete. The more genetically robust narrow leaf width trait was proposed to be used as a reliable phenotypic marker for finding high vein density variants in rice in future screens

Inheritance data for T₄ insertion candidate lines.

<p>*Observed ratio differed from expected ratio at p<0.05. m: mutant phenotype. wt: wild-type phenotype. The control, Tainung 67 wild-type, had an average of 5.02 ± 0.02 veins mm⁻¹ and a maximum of 6 veins mm⁻¹. Vein densities of 6.5 veins mm⁻¹ were considered to be a mutant phenotype. The Tainung 67 wild-type had two out of eighty-eight plants with <7 mm leaf width, one plant with <6 mm leaf width, and no plants with <5 mm leaf width (and an average leaf width of 9.53 <b><i>±</i></b> 0.08 mm). Leaf widths of 6 mm or less were considered to be a mutant phenotype.</p

Frequency distribution of vein densities in the deletion mutant population and their control in the screen.

<p>Values above chart bars denote the number of mutant plants for each high vein density. Distribution frequencies of deletion mutant and control plants were largely the same. Differences were at the extremity of the binomial curve at higher vein densities (930 (1.2%), 112 (0.14%) and 13 (0.02%) mutant plants for respectively 6.5, 7 and 7.5 veins mm⁻¹) where lines were selected as potential candidate mutants for increased vein density.</p

Leaf veins of mutant and wild-type rice lines.

<p>Images of veins within a 2 mm leaf width were captured at 2–3 weeks after sowing. Line E19076-1-5-3 is an M₅ IR64 mutant. Line M0110124-A-2-2 is a T₃ Tainung 67 mutant.</p

Comparing vein density frequencies of IR64 deletion mutants and Tainung 67 insertion mutants.

<p>Frequencies are expressed in percentages of the corresponding mutant population.</p

Frequency distribution of vein densities in the T₁–T₃ insertion mutant populations and their controls.

<p>Values within the chart denote the number of mutant plants for each of the highest vein density values. Distribution frequencies of insertion mutant and wild-type plants were largely the same. Differences were at the extremity of the binomial curve at higher vein densities (eight (0.04%) and 26 (0.12%) mutant plants for respectively 7 and 8 veins mm⁻¹) where lines were selected as potential candidate mutants for altered vein spacing.</p

Phenotype combinations in the F₂ progeny of double mutant, G558-11-5-2/E22097-1-3-1.

<p>n_n: recessive, narrow leaf width allele. N_n: dominant, wild-type leaf width allele. v_n: recessive, high vein density allele. V_n: dominant, wild-type vein density allele. Phenotypes were evaluated at the 5^th–7^th leaf stage except for the vein densities of progeny plants with very narrow leaves. Their vein counts per 2 mm could not be taken at the 5^th–7^th leaf stage so were instead taken at the late vegetative stage. Wild-type leaf width: 8–9 mm. Narrow leaf width: 4–6 mm. Very narrow leaf width: 1–2 mm. Wild-type vein density: ≥5<6.5 veins mm⁻¹. High vein density: ≥6.5<7.5 veins mm⁻¹. Very high vein density: ≥7.5 veins mm⁻¹. Both candidate mutant lines G558-11-5-2 and E22097-1-3-1 had the phenotypic traits of narrow leaves and high vein density. The data characterizes F₂ progeny descended from a single F₁ plant. The F₁ plants all had wild-type vein density and leaf width.</p