Abscission is a universal process whereby plants shed their organs, such as flowers, fruit and leaves. In tomato, the non-allelic mutations jointless and jointless-2 have been discovered as recessive mutations that completely suppress the formation of pedicel abscission zones. A high resolution genetic map of jointless-2 was constructed using 1,122 jointless F2 plants. Restriction fragment length polymorphism (RFLP) marker RPD140 completely co-segregated with the jointless-2 locus and mapped in a 2.4 cM interval between RFLP markers CD22 and TG618. To chromosome walk to jointless-2, all three markers were used to screen a bacterial artificial chromosome (BAC) library and contigs were developed. Intensive efforts to expand and merge the BAC contigs were unsuccessful because of the highly repetitive sequence content on the distal ends of each contig. To determine the physical distance between and the orientation of the three contigs, we used high resolution pachytene fluorescence in situ hybridization (FISH) mapping. The RPD140 contig was positioned in the centromeric region of chromosome 12 between two large pericentric heterochromatin blocks, about 50 Mb from the TG618 contig on the short arm and 10 Mb from the CD22 contig on the long arm, respectively. Based on high resolution genetic and physical mapping, we conclude that the jointless-2 gene is located within or near the chromosome 12 centromere where 1 cM is approximately 25 Mb in length.

Abscission is the mechanism by which plants disconnect unfertilized flowers, ripe fruits, senescent or diseased organs from the plant. In tomato, pedicel abscission is an important agronomic factor that controls yield and post-harvest fruit quality. Two non-allelic mutations, jointless (j) and jointless-2 (j-2), controlling pedicel abscission zone formation have been documented but only j-2 has been extensively used in breeding. J was shown to encode a MADS-box protein. Using a combination of physical mapping and gene expression analysis we identified a positional candidate, Solyc12g038510, associated with j-2 phenotype. Targeted knockout of Solyc12g038510, using CRISPR/Cas9 system, validated our hypothesis. Solyc12g038510 encodes the MADS-box protein SlMBP21. Molecular analysis of j-2 natural variation revealed two independent loss-of-function mutants. The first results of an insertion of a Rider retrotransposable element. The second results of a stop codon mutation that leads to a truncated protein form. To bring new insights into the role of J and J-2 in abscission zone formation, we phenotyped the single and the double mutants and the engineered alleles. We showed that J is epistatic to J-2 and that the branched inflorescences and the leafy sepals observed in accessions harboring j-2 alleles are likely the consequences of linkage drags.

Abscission zones are specialized regions in plants, usually located at the base of most plant parts, such as flowers, fruit and leaves, where organs are shed. Although a great deal of information is known about the physiological and biochemical events that lead to organ shedding, very little is known of the molecular events that lead to the formation of the abscission zone itself. In tomato, two recessive mutations have been discovered that completely suppress the formation of flower and fruit pedicel abscission zones, i.e., jointless (j) and jointless-2 (j-2), both tentatively localized to chromosome 11 about 30 cM apart. Because the study of the control of abscission zone development is important for both basic and applied research we are using a map-based cloning approach to identify the jointless genes. The first step in any positional cloning experiment is to establish segregating mapping populations for the target gene and identify closely linked molecular markers that flank the locus. In this study, bulked segregant analysis was used to identify a RAPD marker associated with the j-2 locus, RPD140. To determine the chromosome location of RPD140, we converted it to an RFLP marker that was then mapped on the Cornell reference tomato map in a marker-dense region of chromosome 12. To verify that the j-2 locus was located on tomato chromosome 12, we used nine chromosome 12 RFLP markers linked with RPD140 to map the j-2 gene in an interspecific F2 mapping population of 151 plants segregating for j-2. The j-2 gene was localized to a 3.0-cM interval between RPD140 and TG618 on tomato chromosome 12.