Tomato POLLEN DEFICIENT 2 encodes a G-type lectin receptor kinase required for viable pollen grain formation.

Pollen development is a crucial biological process indispensable for seed set in flowering plants and for successful crop breeding. However, little is known about the molecular mechanisms regulating pollen development in crop species. This study reports a novel male-sterile tomato mutant, pollen deficient 2 (pod2), characterized by the production of non-viable pollen grains and resulting in the development of small parthenocarpic fruits. A combined strategy of mapping-by-sequencing and RNA interference-mediated gene silencing was used to prove that the pod2 phenotype is caused by the loss of Solanum lycopersicum G-type lectin receptor kinase II.9 (SlG-LecRK-II.9) activity. In situ hybridization of floral buds showed that POD2/SlG-LecRK-II.9 is specifically expressed in tapetal cells and microspores at the late tetrad stage. Accordingly, abnormalities in meiosis and tapetum programmed cell death in pod2 occurred during microsporogenesis, resulting in the formation of four dysfunctional microspores leading to an aberrant microgametogenesis process. RNA-seq analyses supported the existence of alterations at the final stage of microsporogenesis, since we found tomato deregulated genes whose counterparts in Arabidopsis are essential for the normal progression of male meiosis and cytokinesis. Collectively, our results revealed the essential role of POD2/SlG-LecRK-II.9 in regulating tomato pollen development.

The Tomato SlVIPP1 Gene Is Required for Plant Survival Through the Proper Development of Chloroplast Thylakoid Membrane.

Since membranes play essential roles in all living beings, all cells have developed mechanisms for efficient and fast repair of membrane damage. In Escherichia coli, the Phage shock stress A (PspA) protein is involved in the maintenance of the integrity of its inner membrane in response to the damage produced by exposure to stress conditions. A role in thylakoid membrane maintenance and reorganization has been proposed for Vesicle Inducing Protein in Plastid 1 (VIPP1), the putative PspA ortholog in Arabidopsis thaliana. While some membranes of plant cells have been extensively studied, the biosynthesis and maintenance of chloroplast thylakoid membrane remains poorly known. Here, we report the cloning and functional characterization of the tomato (Solanum lycopersicum L.) ortholog of Escherichia coli PspA and Arabidopsis thaliana VIPP1, which we dubbed SlVIPP1. Our genetic and molecular characterization of slvipp1, an insertional mutant, allowed us to conclude that the tomato SlVIPP1 gene is needed for development, as Arabidopsis VIPP1, but not Escherichia coli PspA. Homozygous slvipp1 tomato plants are albino and exhibit early lethality and highly aberrant chloroplast development with almost complete absence of thylakoids. The phenotype of tomato RNAi lines and that of additional slvipp1 alleles generated by CRISPR/Cas9 gene editing technology confirmed that the morphological and histological aberrations shown by slvipp1 homozygotes are caused by VIPP1 lack of function. We also found that tomato SlVIPP1 overexpression does not cause any visible effect on plant morphology and viability. Our work with slvipp1 plants evidences that SlVIPP1 is an essential gene required for tomato survival, since its function is crucial for the proper formation and/or maintenance of thylakoid membranes.

A collection of enhancer trap insertional mutants for functional genomics in tomato.

With the completion of genome sequencing projects, the next challenge is to close the gap between gene annotation and gene functional assignment. Genomic tools to identify gene functions are based on the analysis of phenotypic variations between a wild type and its mutant; hence, mutant collections are a valuable resource. In this sense, T-DNA collections allow for an easy and straightforward identification of the tagged gene, serving as the basis of both forward and reverse genetic strategies. This study reports on the phenotypic and molecular characterization of an enhancer trap T-DNA collection in tomato (Solanum lycopersicum L.), which has been produced by Agrobacterium-mediated transformation using a binary vector bearing a minimal promoter fused to the uidA reporter gene. Two genes have been isolated from different T-DNA mutants, one of these genes codes for a UTP-glucose-1-phosphate uridylyltransferase involved in programmed cell death and leaf development, which means a novel gene function reported in tomato. Together, our results support that enhancer trapping is a powerful tool to identify novel genes and regulatory elements in tomato and that this T-DNA mutant collection represents a highly valuable resource for functional analyses in this fleshy-fruited model species.

Albino T-DNA tomato mutant reveals a key function of 1-deoxy-D-xylulose-5-phosphate synthase (DXS1) in plant development and survival.

Photosynthetic activity is indispensable for plant growth and survival and it depends on the synthesis of plastidial isoprenoids as chlorophylls and carotenoids. In the non-mevalonate pathway (MEP), the 1-deoxy-D-xylulose-5-phosphate synthase 1 (DXS1) enzyme has been postulated to catalyze the rate-limiting step in the formation of plastidial isoprenoids. In tomato, the function of DXS1 has only been studied in fruits, and hence its functional relevance during plant development remains unknown. Here we report the characterization of the wls-2297 tomato mutant, whose severe deficiency in chlorophylls and carotenoids promotes an albino phenotype. Additionally, growth of mutant seedlings was arrested without developing vegetative organs, which resulted in premature lethality. Gene cloning and silencing experiments revealed that the phenotype of wls-2297 mutant was caused by 38.6 kb-deletion promoted by a single T-DNA insertion affecting the DXS1 gene. This was corroborated by in vivo and molecular complementation assays, which allowed the rescue of mutant phenotype. Further characterization of tomato plants overexpressing DXS1 and comparative expression analysis indicate that DXS1 may play other important roles besides to that proposed during fruit carotenoid biosynthesis. Taken together, these results demonstrate that DXS1 is essentially required for the development and survival of tomato plants.

Uncovering the genetic architecture of Colletotrichum lindemuthianum resistance through QTL mapping and epistatic interaction analysis in common bean.

Colletotrichum lindemuthianum is a hemibiotrophic fungal pathogen that causes anthracnose disease in common bean. Despite the genetics of anthracnose resistance has been studied for a long time, few quantitative trait loci (QTLs) studies have been conducted on this species. The present work examines the genetic basis of quantitative resistance to races 23 and 1545 of C. lindemuthianum in different organs (stem, leaf and petiole). A population of 185 recombinant inbred lines (RIL) derived from the cross PMB0225 × PHA1037 was evaluated for anthracnose resistance under natural and artificial photoperiod growth conditions. Using multi-environment QTL mapping approach, 10 and 16 main effect QTLs were identified for resistance to anthracnose races 23 and 1545, respectively. The homologous genomic regions corresponding to 17 of the 26 main effect QTLs detected were positive for the presence of resistance-associated gene cluster encoding nucleotide-binding and leucine-rich repeat (NL) proteins. Among them, it is worth noting that the main effect QTLs detected on linkage group 05 for resistance to race 1545 in stem, petiole and leaf were located within a 1.2 Mb region. The NL gene Phvul.005G117900 is located in this region, which can be considered an important candidate gene for the non-organ-specific QTL identified here. Furthermore, a total of 39 epistatic QTL (E-QTLs) (21 for resistance to race 23 and 18 for resistance to race 1545) involved in 20 epistatic interactions (eleven and nine interactions for resistance to races 23 and 1545, respectively) were identified. None of the main and epistatic QTLs detected displayed significant environment interaction effects. The present research provides essential information not only for the better understanding of the plant-pathogen interaction but also for the application of genomic assisted breeding for anthracnose resistance improvement in common bean through application of marker-assisted selection (MAS).

Genetic analysis of single-locus and epistatic QTLs for seed traits in an adapted × nuña RIL population of common bean (Phaseolus vulgaris L.).

KEY MESSAGE: The QTLs analyses here reported demonstrate the significant role of both individual additive and epistatic effects in the genetic control of seed quality traits in the Andean common bean. Common bean shows considerable variability in seed size and coat color, which are important agronomic traits determining farmer and consumer acceptability. Therefore, strategies must be devised to improve the genetic base of cultivated germplasm with new alleles that would contribute positively to breeding programs. For that purpose, a population of 185 recombinant inbred lines derived from an Andean intra-gene pool cross, involving an adapted common bean (PMB0225 parent) and an exotic nuña bean (PHA1037 parent), was evaluated under six different--short and long-day--environmental conditions for seed dimension, weight, color, and brightness traits, as well as the number of seed per pod. A multi-environment Quantitative Trait Loci (QTL) analysis was carried out and 59 QTLs were mapped on all linkage groups, 18 of which had only individual additive effects, while 27 showed only epistatic effects and 14 had both individual additive and epistatic effects. Multivariate models that included significant QTL explained from 8 to 68 % and 2 to 15 % of the additive and epistatic effects, respectively. Most of these QTLs were consistent over environment, though interactions between QTLs and environments were also detected. Despite this, QTLs with differential effect on long-day and short-day environments were not found. QTLs identified were positioned in cluster, suggesting that either pleiotropic QTLs control several traits or tightly linked QTLs for different traits map together in the same genomic regions. Overall, our results show that digenic epistatic interactions clearly play an important role in the genetic control of seed quality traits in the Andean common bean.

Marker-based linkage map of Andean common bean (Phaseolus vulgaris L.) and mapping of QTLs underlying popping ability traits.

BACKGROUND: Nuña bean is a type of ancient common bean (Phaseolus vulgaris L.) native to the Andean region of South America, whose seeds possess the unusual property of popping. The nutritional features of popped seeds make them a healthy low fat and high protein snack. However, flowering of nuña bean only takes place under short-day photoperiod conditions, which means a difficulty to extend production to areas where such conditions do not prevail. Therefore, breeding programs of adaptation traits will facilitate the diversification of the bean crops and the development of new varieties with enhanced healthy properties. Although the popping trait has been profusely studied in maize (popcorn), little is known about the biology and genetic basis of the popping ability in common bean. To obtain insights into the genetics of popping ability related traits of nuña bean, a comprehensive quantitative trait loci (QTL) analysis was performed to detect single-locus and epistatic QTLs responsible for the phenotypic variance observed in these traits. RESULTS: A mapping population of 185 recombinant inbred lines (RILs) derived from a cross between two Andean common bean genotypes was evaluated for three popping related traits, popping dimension index (PDI), expansion coefficient (EC), and percentage of unpopped seeds (PUS), in five different environmental conditions. The genetic map constructed included 193 loci across 12 linkage groups (LGs), covering a genetic distance of 822.1 cM, with an average of 4.3 cM per marker. Individual and multi-environment QTL analyses detected a total of nineteen single-locus QTLs, highlighting among them the co-localized QTLs for the three popping ability traits placed on LGs 3, 5, 6, and 7, which together explained 24.9, 14.5, and 25.3% of the phenotypic variance for PDI, EC, and PUS, respectively. Interestingly, epistatic interactions among QTLs have been detected, which could have a key role in the genetic control of popping. CONCLUSIONS: The QTLs here reported constitute useful tools for marker assisted selection breeding programs aimed at improving nuña bean cultivars, as well as for extending our knowledge of the genetic determinants and genotype x environment interaction involved in the popping ability traits of this bean crop.