RFLP studies of genetic relationships among inbred lines of the cultivated sunflower, Helianthus annuus L.: evidence for distinct restorer and maintainer germplasm pools

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Abstract
One-hundred-and-eighty-one nuclear DNA probes were used to examine restriction-fragment length polymorphism in inbred lines of the cultivated sunflower (Helianthus annuus L.). The probes were from six libraries: two genomic libraries — one made with PstI and the other with HindIII, and four cDNA libraries — from etiolated plantlets, green leaves, ovaries, petals and anthers. Total DNA from 17 inbred lines representing an overview of the genetic stocks of sunflower, including restorer and maintainer lines of the classical cytoplasmic male sterility, was digested with four different restriction enzymes and probed in 331 probe-enzyme combinations. Of 181 clones analysed, 73 probes were found to be polymorphic. Genetic distances between inbreds were calculated from the resultant proportion of shared bands and submitted to principal component analysis and the UPGMA ‘tree-making’ method. The RFLP analysis allowed a clear differentiation between restorer and maintainer lines of the cytoplasmic male sterility, together with a grouping of some of the genotypes from the same origin. The analysis of the accuracy of distance estimation as a function of the number of probe-enzyme combinations used, indicates that 40–50 combinations ensure a confidence level of near 95%. Considering the inbreds as representatives of the range of cultivated inbreds, estimates of gene diversity, as well as estimates of average gene diversity between and within the sets of restorer and maintainer lines, were calculated. Estimation of gene diversity showed that the available genetic variability in cultivated sunflower, based on allelic frequencies, is lower than that of other plants (H=0.20). Moreover, we show that the proportion of genetic variability due to the difference between maintainer and restorer lines (Dm) is about 2%.