The role of Pv08 in the original domestication of Middle American common beans also cannot yet be ruled out. Our Pv03 QTL mapping peak in the MxB population was closer in physical distance to PvPdh1 than what has been previously identified through QTL mapping in a different recombinant inbred population—ICA Bunsi x SXB405 . These results are still not as close to the gene as those achieved by GWAS with a much larger SNP dataset . The correlation between PvPdh1 allele and pod twists indicates that the gene may modify the twisting force of pod walls. This has been seen in the soybean ortholog as well as across numerous legume species . The complex dominance of PvPdh1-mediated shattering resistance parallels the pattern seen in soybean pods, in which the phenotyping method affects the pattern of dominance . The desiccation method was faster to phenotype than counting pod twists and also produced results which were much more correlated with genotype. This indicates that the desiccator method may be a more effective method of phenotypic screening than counting twists. The recessive nature of pod shattering when phenotyped by the desiccator method means that carriers of the resistant Pvpdh1 allele may demonstrate high levels of pod shattering in early breeding program generations because of heterozygosity and should not be eliminated without direct genetic evaluation or subsequent progeny tests. Further,how to set up a vertical farm the recessive nature of shattering resistance when phenotyped by the desiccator method also indicates that recurrent back crossing based on phenotyping alone would not be practical for the trait.
Pvpdh1 therefore requires a genetic marker for screening of progenies that carry the shattering-resistance allele.Our results indicate that durable resistance to pod shattering has evolved independently in both the Middle American and Andean gene pools of common bean. Despite this, many varieties in both gene pools continue to display the wild type propensity to shattering, and this is strongly associated with market class. Our results agree with earlier anecdotal observations that pod shattering is most problematic in the black and cranberry market classes , the two categories with the highest rates of pod shattering in the MDP and ADP. In contrast, market classes with the lowest rates of pod shattering are those in which the resistant Pvpdh1 allele is most abundant . While direct comparisons between the Andean and Middle American gene pools are complicated by the fact that the populations were grown in different years, the desiccation treatment used to induce pod fracture was identical between populations. In any case, it is clear that many varieties of both gene pools experience high levels of pod shattering and would benefit from the introgression of shattering-resistance alleles. Market demands require most new varieties of common bean to conform to standards for several complex traits, such as seed size, shape, color, leading most modern breeding to focus preferentially on intra-race crosses. Marker assisted back crossing would greatly facilitate the transfer of the shattering-resistant allele into other ecogeographic races, while maintaining the complex genetic background required in a market class. A better understanding of the PvPdh1 locus, as well as molecular markers associated with it, will become increasingly important for crop improvement as conditions become more arid in the twenty-first century.Our haplotype diversity results are consistent with the hypothesis that there has been stronger selection pressure on PvPdh1 in race Durango than in race Mesoamerica.
After selection of the shattering resistant allele at PvPdh1, race Durango types differentiated into just two additional new haplotypes, which represent 3% of the group’s sampled varieties. The non-shattering character found in the low-frequency haplotypes indicates that these groups may have differentiated since the mutation in PvPdh1, rather than being ancestral reflicts of a shattering-susceptible race Durango progenitor. In contrast, race Mesoamerica includes six total haplotypes, and the ive least common of these together represent 7% of the sampled varieties. This is more than double the frequency of minor haplotypes than in race Durango. These less-common variants could be the subject of future study to identify whether secondary mutations in PvPdh1 have independently arisen to regulate pod shattering in a subset of varieties within race Mesoamerica.While EcoRI is a highly stable, robust enzyme, TaqII is a high molecular weight, lower-stability enzyme, which requires highly specific conditions for optimal DNA cleavage . This includes a predigestion PCR product cleanup and extreme care in handling of the enzyme. Although TaqII treatment always led to digestion of susceptible alleles, this digestion was sometimes only partial, leading to ambiguity between homozygous susceptible and heterozygous individuals. Further, the cleavage of shattering-susceptible alleles is generally less desirable than cleavage of resistant alleles to reduce the risk of selecting susceptible types due to technical errors. While the TaqII-based marker may be ideal for initial parental screening, the tightly linked EcoRI-based CAPS marker may be more practical for rapid, efficient screening of large breeding populations. The CAPS markers developed here may be valuable for rapidly transferring the pod-shattering resistance of race Durango into the market classes of race Mesoamerica and the Andean gene pool. Pod shattering is a complex quantitative trait and is regulated by multiple alleles and environmental variables. Indeed, selection based on phenotyping alone will not always be predictive of an individual’s susceptibility to pod shattering , leading to imperfect selection accuracy.
Our CAPS markers will provide a more accurate and rapid method to genetically evaluate an individual’s resistance to pod shattering. Similarly, the SNPs used to develop these CAPS markers could be converted to Kompetitive Allele specific PCR markers through commercially available services. Phenotypically, this trait cannot be measured until after plants have fully senesced, delaying selection and requiring breeders to invest heavily in non-desired plants. Further, it often requires additional heat treatment incubation periods or labor-intensive analyses with specialized equipment, such as mechanical force measurement gauges, to study accurately . In contrast, our genetic tests can be conducted rapidly on segregating populations of seedlings, reducing costs for breeding programs and hastening genetic improvement. These markers will also allow breeders to accurately pyramid shattering resistance alleles from the Andean and Middle American gene pools for the first time, potentially leading to stronger resistance to pod shattering than what is provided by Pvpdh1 alone. In turn, this will facilitate the development of varieties that are more tolerant of warm, dry environmental conditions where pod shattering is most problematic.Tolerance to desiccation can be defined as the ability of an organism to survive extreme dehydration and then to resume normal growth upon rehydration.Vascular plants with the ability to revive from extreme desiccation are commonly called as resurrection plants.3 To date, more than 300 angiosperms,what is vertical growing including a few dicotyledonous plants, have been identified as resurrection plants.Tolerance to desiccation in resurrection plants is a complex process that involves many physiological and metabolic mechanisms.The strategies adopted by resurrection plants to survive desiccation may resemble those seen in seeds.Water deficit is initially accompanied by osmotic adjustment to protect against cellular damage, which is accomplished by the accumulation of large amounts of sugars, amino acids, and small polypeptides such as late embryogenesis abundant proteins and dehydrins. Increasing water deficit results in the activation of mechanisms to cope with desiccation-induced structural and functional alterations of macromolecules and membranes, the accumulation of toxic substances and free radicals, and mechanical damage associated with the loss of turgor.Under desiccation stress, the oxidative damage caused by the production of reactive oxygen species is enhanced, especially in chloroplasts. Two different strategies are adopted by resurrection plants to minimize ROS damage. Homoiochlorophyllous resurrection plants conserve the structure of their photosynthetic apparatus, synthesize anthocyanins, and increase the activity of antioxidants during desiccation, whereas poikilochlorophyllous resurrection plants demolish chloroplast pigments and membranes, thus reducing cellular sources of ROS.6 Resurrection plants are excellent models for studying molecular mechanisms that could potentially serve as biotechnological tools for enhancing drought tolerance.There have been many studies of the molecular effects of drought on model plants such as Arabidopsis and rice,but information is scarce on the desiccation tolerance mechanisms of resurrection plants.Transcriptomics has been used to generate global gene expression atlases, such as for powdery mildew resistance,horticultural traits in apple,fruit ripening, and cherry fruit development.To understand desiccation tolerance mechanisms, transcriptomic and proteomic studies have been performed in resurrection plants such as Sporobolus stapfianus, Tortula ruralis, Xerophyta viscosa,and Boea hygrometrica.However, large-scale expressed sequence tag sequencing analysis using high-throughput sequencing and de novo assembly strategies have only been conducted in two herbaceous species, Craterostigma plantagineum and Haberlea rhodopensis.
Myrothamnus flabellifolia, a woody homoiochlorophyllous resurrection plant growing in the mountainous regions of central and southern Africa,is probably the most primitive angiosperm to show extreme tolerance to desiccation.It displays novel anatomical, ultra-structural, and biochemical adaptations to desiccation stress and has been the subject of a number of physiological and biochemical studies. Under severe drought conditions, M. flabellifolia leaves can become air-dry, folding in a unique fan-like manner.Upon rewatering, the leaves return to their original color and shape within 24 hours.Biochemical studies of the cell walls have demonstrated an abundance of arabinose polymer side chains, consisting of arabinans associated with the pectin matrix, which may explain the flexibility of the mesophyll cells, allowing leaf morphology to be rapidly recovered after rehydration.During desiccation, presumably to protect from ROS, the leaves synthesize large amounts of anthocyanins and change their color from green to dull-brown.High levels of phenols, including tannins, arbutin, and tri-O-galloylquinic acid, have been reported in the leaves of M. flabellifolia. Differences in phenolic content and composition may be associated with differences in tolerance to desiccation stress among populations of M. flabellifolia.Moreover, desiccated leaves of M. flabellifolia contain high levels of saccharides, although the precise constituents differ.The molecular mechanisms underlying the tolerance of M. flabellifolia and its ability to rapidly rehydrate are still largely unknown. In the present work, we attempt to investigate the transcriptome dynamics in response to dehydration and rehydration in M. flabellifolia. Most woody fruit and ornamental plants can encounter extreme drought stress. Therefore, understanding this plant’s extreme tolerance to desiccation and drought can aid the development of strategies for improving drought stress resistance in horticultural crops.To evaluate the potential functions of genes that showed significant transcriptional changes during dehydration and rehydration in M. flabellifolia, we examined the GO enrichment of DTGs at each dehydration and rehydration stage. The biological processes that were significantly enriched early in dehydration included many defense responses such as response to chitin, cold, wounding, and fungi . This is consistent with a previous transcriptomic study in the resurrection plant H. rhodopensis in which many genes involved in acquiring tolerance to a variety of abiotic and biotic stresses were induced by desiccation stress.In striking contrast, the DTGs represented in moderately dehydrated and desiccated leaves participate in photosystem II assembly, thylakoid membrane organization, chlorophyll biosynthesis, and photosynthesis . These data suggest that alteration of photosynthesis is an important strategy in response to desiccation for M. flabellifolia. Moreover, genes involved in isopentenyl diphosphate biosynthesis were enriched in moderately dehydrated, desiccated, and rehydrated leaves . Isopentenyl disphosphate is a precursor of all plant isoprenoids,which are a diverse group of metabolites including primary metabolites such as sterols, chlorophylls, carotenoids, quinones, and hormones and secondary metabolites participating in plant defense and communication such as pigments, volatiles, and defense compounds.To further delineate the metabolic pathways participating in the dehydration and rehydration responses, we mapped the DTGs into the Kyoto Encyclopedia of Genes and Genomes database. A total of 23 and 18 biochemical pathways were significantly enriched during dehydration and rehydration, respectively . During dehydration, consistent with the GO enrichmentanalysis, the enriched biochemical pathways involved diverse metabolites, including secondary metabolites, starch, sucrose, fatty acids, fructose and mannose, and photosynthesis, suggesting a global response to desiccation in M. flabellifolia. As one example, the flavonoid biosynthesis pathway was significantly enriched during dehydration, consistent with the role of anthocyanins, a flavonoid class, in the ‘green-to-brown’/‘brown-to-green’ leaf color alternation during dehydration and rehydration.During rehydration, photosynthetic genes were significantly enriched . This is consistent with a previous study in M. flabellifolia showing that photosynthesis resumes rapidly, reaching full capacity within three days of rehydration.The recovery of vital functions and metabolic activities during rehydration is indicated by alterations of genes encoding a range of structural and physiological processes.For example, genes encoding enzymes involved in ribosome and aminoacyl-rRNA biosynthesis, essential for the re-establishment of normal cellular metabolism, are enriched during rehydration .