Localization of QTLs for in vitro plant regeneration in tomato

Abstract

Background: Low regeneration ability limits biotechnological breeding approaches. The influence of genotype in the regeneration response is high in both tomato and other important crops. Despite the various studies that have been carried out on regeneration genetics, little is known about the key genes involved in this process. The aim of this study was to localize the genetic factors affecting regeneration in tomato.

Results: We developed two mapping populations (F2 and BC1) derived from a previously selected tomato cultivar (cv. Anl27) with low regeneration ability and a high regeneration accession of the wild species Solanum pennellii (PE-47). The phenotypic assay indicated dominance for bud induction and additive effects for both the percentage of explants with shoots and the number of regenerated shoots per explant. Two linkage maps were developed and six QTLs were identified on five chromosomes (1, 3, 4, 7 and 8) in the BC1 population by means of the Interval Mapping and restricted Multiple QTL Mapping methods. These QTLs came from S. pennellii, with the exception of the minor QTL located on chromosome 8, which was provided by cv. Anl27. The main QTLs correspond to those detected on chromosomes 1 and 7. In the F2 population, a QTL on chromosome 7 was identified on a similar region as that detected in the BC1 population. Marker segregation distortion was observed in this population in those areas where the QTLs of BC1 were detected. Furthermore, we located two tomato candidate genes using a marker linked to the high regeneration gene: Rg-2 (a putative allele of Rg-1) and LESK1, which encodes a serine/threonine kinase and was proposed as a marker for regeneration competence. As a result, we located a putative allele of Rg-2 in the QTL detected on chromosome 3 that we named Rg-3. LESK1, which is also situated on chromosome 3, is outside Rg-3. In a preliminary exploration of the detected QTL peaks, we found several genes that may be related to regeneration.

Conclusions: In this study we have identified new QTLs related to the complex process of regeneration from tissue culture. We have also located two candidate genes, discovering a putative allele of the high regeneration gene Rg-1 in the QTL on chromosome 3. The identified QTLs could represent a significant step toward the understanding of this process and the identification of other related candidate genes. It will also most likely facilitate the development of molecular markers for use in gene isolation.

Figure 1

Population distributions for regeneration traits. a) The percentage of explants with buds (B), b) The percentage of explants with plants (R) and c) The percentage of plants per explant with shoots (PR). The F₂ population (dark) is derived from selfing an F₁, the result of a cross between the tomato cv. Anl27 (P1) and S. pennellii PE-47 (P2). The BC₁ population (grey) is the result of crossing the tomato cv. Anl27 and the F₁ plants. Maternal (P1), Paternal (P2), F₁, F₂ and BC₁ mean values are indicated by arrows.

Figure 2

a) Tomato genetic linkage map of F₂ population derived from S. lycopersicum (cv. Anl27) × S. pennellii (PE-47) and QTLs detected for regeneration traits by IM. b) Tomato genetic linkage map of BC₁ population derived from S. lycopersicum (cv. Anl27) × F₁ (cv. Anl27 × PE-47) and QTLs detected for regeneration traits by rMQM. The segregated data were classified into 12 linkage groups, which corresponded to the Tomato-EXPEN 2000 map; italics indicate markers with segregation significantly skewed (P < 0.05) in favour of parent alleles. The colors specify the direction of the segregation distortion (red: markers skewed toward the alleles of cultivated tomato; green: markers skewed toward the alleles of the wild parent). Green bars reflect QTLs from S. pennellii: SpRg-1, Rg-3, SpRg-4a, SpRg-4b and SpRg-7; the red bar reflects the SlRg-8 QTL from S. lycopersicum. Regeneration traits: B (Bud percentage), R (Regeneration percentage) and PR (Productivity rate). The black star labels the acid invertase gene (inv^penn) mapped on chromosome 3 included in the Rg-3 QTL range.