Molecular breeding in developing countries: challenges and perspectives.

Molecular breeding (MB) holds great promise for developing countries. However, the developing countries are hardly homogeneous in its implementation. Whilst newly industrialised countries routinely use several MB applications and are exploring the latest approaches, developing countries with mid-level economies are testing marker applications and taking initial steps towards adopting MB in day-to-day breeding. Various bottlenecks still impede adoption in these countries. Limited human resources and inadequate field infrastructure remain major challenges, although through virtual platforms aided by the information and communication technology revolution, breeders now have better access to genomic resources, advanced laboratory services, and robust analytical and data management tools. These developments are bound to have impact crop improvement in developing countries.

Construction of a reference linkage map for melon.

A map of melon (Cucumis melo L.) with 411 markers (234 RFLPs, 94 AFLPs, 47 RAPDs, 29 SSRs, five inter-SSRs, and two isozymes) and one morphological trait (carpel number) was constructed using the F2 progeny of a cross between the Korean accession P1161375 and the Spanish melon type 'Pinyonet Piel de Sapo'. RFLPs were obtained using 212 probes from different genomic and cDNA melon libraries, including 16 Arabidopsis ESTs, 13 Cucumis known genes, and three resistant gene homologues. Most loci (391) mapped to 12 major linkage groups, spanning a total genetic distance of 1197 cM, with an average map interval of 3 cM/marker. The remaining 21 loci (six RAPDs and 15 AFLPs) were not linked. A majority (66%) of the markers were codominant (RFLPs, SSRs, and isozymes), making them easily transferable to other melon crosses. Such markers can be used as a reference, to merge other melon and cucumber maps already constructed. Indeed, some of them (23 SSRs, 14 RFLPs, one isozyme, and one morphological trait) could act as anchor points with other published cucurbit maps.

Development of a second generation linkage map for almond using RAPD and SSR markers.

Fifty-four RAPD (random amplified polymorphic DNA) markers and 6 SSRs (simple sequence repeats) were included in a molecular marker map with 120 RFLPs (restriction fragment length polymorphisms) and 7 isozyme genes previously constructed using the offspring of a cross between the almond (Prunus amygdalus) cultivars 'Ferragnès' and 'Tuono'. Only highly reproducible RAPDs segregating 1:1 were used. To identify these markers, a total of 325 primers were screened, from which 41 produced RAPDs useful for mapping. Polymorphism was detected in six of the eight Prunus SSRs (simple sequence repeats) studied, thus enabling these to be mapped. All markers were placed on the 8 linkage groups previously identified. The number of new markers included in the map of 'Ferragnès' was 33 for a total of 126, and 30 in the map of 'Tuono' for a total of 99. The sizes of the maps of 'Ferragnès' (415 cM) and 'Tuono' (416 cM) were similar, representing a 5% increase over the maps constructed solely with isozymes and RFLPs. The estimated total size of the almond map was of 457 cM. Some markers were placed in zones with low density of markers and others in the extreme of linkage groups. The use of RAPD markers to complete genetic maps constructed with transferable markers is discussed.

Developing SCAR markers to study predation on Trialeurodes vaporariorum.

DNA markers of Trialeurodes vaporariorum were developed to detect remains of these whitefly in the gut of the predator Dicyphus tamaninii. A 2400-bp DNA fragment of T. vaporariorum, absent in other closely related prey species and in the predator banding pattern, was identified by random amplified polymorphic DNA (RAPD) analysis. After cloning and sequencing this fragment, two pairs of sequence-characterized amplified region (SCAR) primers were developed, amplifying single bands of 2100 bp and 310 bp, respectively. Detection of T. vaporariorum DNA in the predator gut was only possible using the primers that amplified the shortest fragment. Specificity tests performed with this pair of primers showed the presence of the 310-bp band for T. vaporariorum in all stages.

A linkage map with RFLP and isozyme markers for almond.

Inheritance and linkage studies were conducted with seven isozyme genes and 120 RFLPs in the F1 progeny of a cross between almond cultivars 'Ferragnes' and 'Tuono'. RFLPs were detected using 57 genomic and 43 cDNA almond clones. Eight of the cDNA probes corresponded to known genes (extensin, prunin (2), α-tubulin, endopolygalacturonase, oleosin, actin depolymerizing factor and phosphoglyceromutase). Single-copy clones were found more frequently in the cDNA (65%) than in the genomic libraries (26%). Two maps were elaborated, one with the 93 loci heterozygous in 'Ferragnes' and another with the 69 loci heterozygous in 'Tuono'. Thirty-five loci were heterozygous in both parents and were used as bridges between both maps. Most of the segregations (91%) were of the 1∶1 or 1∶1∶1∶1 types, and data were analyzed as if they derived from two backcross populations. Eight linkage groups covering 393 cM in 'Ferragnes' and 394 in 'Tuono' were found for each map. None of the loci examined in either map was found to be unlinked. Distorted segregation ratios were mainly concentrated in two linkage groups of the 'Ferragnes' map.

Organisation of the tomato polyphenol oxidase gene family.

We report the isolation and characterization of seven nuclear genes encoding polyphenol oxidase (PPO) in tomato (Lycopersicon esculentum cv. VFNT Cherry). The seven genes (PPOs A, A', B, C, D, E and F) fall into three structural classes (I, II, and III) based on Eco RI and Hind III restriction fragment length polymorphisms (RFLP). RFLP mapping and PFGE analysis demonstrated that the genes reside on chromosome 8, and may be clustered within a 165 kb region. Phage insert mapping demonstrated PPO E and PPO F (both class III), and PPOs B, D and A (classes I, II and I respectively) are grouped within separate 12.4 kb clusters. The complete nucleotide sequence was determined for each gene. Comparison to cDNAs revealed that the PPOs lack introns. A transcript of about 2 kb is expected for each PPO. Each PPO possesses a region encoding a transit peptide characteristic of polypeptides targeted to the thylakoid lumen. Predicted precursor polypeptides range in mass from 66 to 71 kDa and predicted mature polypeptides range from 57 to 62 kDa. All the PPOs encode two putative copper-binding sites characteristic of bacterial, fungal and mammalian tyrosinases. Five of the seven PPOs possess divergent DNA sequences in their 5' promoter regions. These flanking sequence differences may regulate the differential expression of PPO genes.

High density molecular linkage maps of the tomato and potato genomes.

High density molecular linkage maps, comprised of more than 1000 markers with an average spacing between markers of approximately 1.2 cM (ca. 900 kb), have been constructed for the tomato and potato genomes. As the two maps are based on a common set of probes, it was possible to determine, with a high degree of precision, the breakpoints corresponding to 5 chromosomal inversions that differentiate the tomato and potato genomes. All of the inversions appear to have resulted from single breakpoints at or near the centromeres of the affected chromosomes, the result being the inversion of entire chromosome arms. While the crossing over rate among chromosomes appears to be uniformly distributed with respect to chromosome size, there is tremendous heterogeneity of crossing over within chromosomes. Regions of the map corresponding to centromeres and centromeric heterochromatin, and in some instances telomeres, experience up to 10-fold less recombination than other areas of the genome. Overall, 28% of the mapped loci reside in areas of putatively suppressed recombination. This includes loci corresponding to both random, single copy genomic clones and transcribed genes (detected with cDNA probes). The extreme heterogeneity of crossing over within chromosomes has both practical and evolutionary implications. Currently tomato and potato are among the most thoroughly mapped eukaryotic species and the availability of high density molecular linkage maps should facilitate chromosome walking, quantitative trait mapping, marker-assisted breeding and evolutionary studies in these two important and well studied crop species.

Genome-wide reduction in recombination of backcross progeny derived from male versus female gametes in an interspecific cross of tomato.

We have determined that meiotic recombination differs between male and female gametes derived from the same plant. A single F1 plant was backcrossed to each of the parents, Lycopersicon esculentum and L pennellii, as the male (BCE) and female (BCP) parent, respectively. A total of 85 RFLP markers, covering more than 75% of the tomato genome, was used to construct a genetic map for both populations. Since both recurrent parents were homozygous, recombination measured in each population reflects crossing-over rates leading to male (BCE) and female (BCP) gametes. Comparisons were made by interval (genetic distance between two adjacent markers), by chromosome, and for the total length of the genome. Significantly less recombination was observed for male gametes at all levels. No significant relationship was found between areas of reduced recombination and approximate location to the centromere. That selection plays some role could not be eliminated, but no clear evidence was observed for single-locus selection as a major factor in the general reduction of crossing-overs in male gametes.

High resolution RFLP map around the root knot nematode resistance gene (Mi) in tomato.

In the 1940's the root-knot nematode resistance gene (Mi) was introgressed into the cultivated tomato from the wild species, L. peruvianum, and today it provides the only form of genetic resistance against this pathogen. We report here the construction of a high resolution RFLP map around the Mi gene that may aid in the future cloning of this gene via chromosome walking. The map covers the most distal nine map units of chromosome 6 and contains the Mi gene, nine RFLP markers, and one isozyme marker (Aps-1). Based on the analysis of more than 1,000 F2 plants from four crosses, we were able to pinpoint the Mi gene to the interval between two of these markers - GP79 and Aps-1. In crosses containing the Mi gene, this interval is suppressed in recombination and is estimated to be 0.4 cM in length. In contrast, for a cross not containing Mi, the estimated map distance is approximately 5 times greater (ca. 2 cM).Using RFLP markers around Mi as probes, it was possible to classify nematode resistant tomato varieties into three types based on the amount of linked peruvianum DNA still present. Two of these types (representing the majority of the varieties tested) were found to still contain more than 5 cM of peruvianum chromosome - a result that may explain some of the negative effects (e.g. fruit cracking) associated with nematode resistance. The third type (represented by a single variety) is predicted to carry a very small segment of peruvianum DNA (<2 cM) and may be useful in the identification of additional markers close to Mi and in the orientation of clones during a chromosome walk to clone the gene.