Marie-Christine Daunay

QTL analysis of morphological traits in eggplant and implications for conservation of gene function during evolution of solanaceous species

Abstract. An interspecific F2 population from a cross between cultivated eggplant, Solanum melongena, and its wild relative, S. linnaeanum, was analyzed for quantitative trait loci (QTL) affecting leaf, flower, fruit and plant traits. A total of 58 plants were genotyped for 207 restriction fragment length polymorphism (RFLP) markers and phenotyped for 18 characters. One to eight loci were detected for each trait with a total of 63 QTL identified. Overall, 46% of the QTL had allelic effects that were the reverse of those predicted from the parental phenotypes. Wild alleles that were agronomically superior to the cultivated alleles were identified for 42% of the QTL identified for flowering time, flower and fruit number, fruit set, calyx size and fruit glossiness. Comparison of the map positions of eggplant loci with those for similar traits in tomato, potato and pepper revealed that 12 of the QTL have putative orthologs in at least one of these other species and that putative orthology was most often observed between eggplant and tomato. Traits showing potential orthology were: leaf length, shape and lobing; days to flowering; number of flowers per inflorescence; plant height and apex, leaf and stem hairiness. The functionally conserved loci included a major leaf lobing QTL (llob6.1) that is putatively orthologous to the potato leaf (c) and/or Petroselinum (Pts) mutants of tomato, two flowering time QTL (dtf1.1, dtf2.1) that also have putative counterparts in tomato and four QTL for trichomes that have potential orthologs in tomato and potato. These results support the mounting evidence of conservation of gene function during the evolution of eggplant and its relatives from their last common ancestor and indicate that this conservation was not limited to domestication traits.

Genome-Enabled Phylogeographic Investigation of the Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 and Screening for Sources of Resistance Against Its Core Effectors

Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of 17 isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a Select Agent in the United States, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and tended to carry ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of 31 type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Toward this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned, and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.

The Cucurbitaceae and Solanaceae illustrated in medieval manuscripts known as the Tacuinum Sanitatis

BACKGROUND AND AIMS Beginning in the last two decades of the 14th century, richly illuminated versions of the Tacuinum Sanitatis, the Latin translation of an 11th-century Arabic manuscript known as Taqwim al-Sihha bi al-Ashab al-Sitta, were produced in northern Italy. These illustrated manuscripts provide a window on late medieval life in that region by containing some 200 full-page illustrations, many of which vividly depict the harvest of vegetables, fruits, flowers, grains, aromatics and medicinal plants. Our objective was to search for and identify the images of taxa of Cucurbitaceae and Solanaceae. METHODS We have located all reported illustrated Tacuinum Sanitatis and similar or related manuscripts, searched through printed or electronic reproductions of them, categorized six of them that display full-page illustrations as archetypic, and established the identity of the Cucurbitaceae and Solanaceae appearing in these six manuscripts. KEY RESULTS AND CONCLUSIONS Of the Cucurbitaceae, Cucumis sativus (short-fruited cucumbers), Cucumis melo (including round as well as elongate melons), Citrullus lanatus (both sweet watermelons and citrons), and Lagenaria siceraria (including bottle-shaped as well as long gourds), are illustrated. Of the Solanaceae, Solanum melongena (egg-shaped purple aubergines) and Mandragora sp. (mandrake) are illustrated. These depictions include some of the earliest known images of cucumber, casaba melon (Cucumis melo Inodorous Group) and aubergine, each of which closely resembles an extant cultivar-group or market type. Overall, the botanically most accurate images are in the version of the Tacuinum located in the Osterreichische Nationalbibliothek, Vienna, cod. ser. n. 2644. Similarities and differences in botanical accuracy among the images of Cucurbitaceae and Solanaceae in the six archetypal Tacuinum manuscripts suggest to us that another illustrated Tacuinum, now lost, may have antedated and served as a model or inspiration for the six surviving archetypic manuscripts.

Occidental diffusion of cucumber (Cucumis sativus) 500–1300 CE: two routes to Europe

BACKGROUND The cucumber, Cucumis sativus, is one of the most widely consumed fruit vegetables the world over. The history of its dispersal to the Occident from its centre of origin, the Indian subcontinent, has been incorrectly understood for some time, due to the confusion of cucumbers with vegetable melons. Iconographic and literary evidence has shown that cucumber was absent in Roman times, up to 500 CE, but present in Europe by late medieval times, 1300. The objective of the present investigation was to determine more accurately when the cucumber arrived in Europe and by what route. FINDINGS AND CONCLUSIONS The evidence for the movement of C. sativus westward is entirely lexicographical until the 10th century. Syriac, Persian and Byzantine Greek sources suggest the presence of cucumbers, to the east and north-east of the Mediterranean Sea (modern Iran, Iraq and Turkey), by the 6th or 7th century. Arabic medical writings suggest the presence of cucumbers in Spain as early as the mid-9th century and in Tunisia by the early 10th century. Descriptive evidence in Arabic establishes the presence of cucumbers in Andalusia by the second half of the 10th century. Latin translations from Arabic sources indicate the presence of cucumbers in southern Italy by the second half of the 11th century. These writings, together with lexicographical discrepancies in names of cucurbits in late medieval Latin writings, suggest that cucumber was introduced to Europe by two independent diffusions. One diffusion appears to have been overland from Persia into eastern and northern Europe and preceded the Islamic conquests. The other, subsequent diffusion into western and southern Europe, was probably by a mostly maritime route from Persia or the Indian subcontinent into Andalusia.

The potential for crop to wild hybridization in eggplant (Solanum melongena; Solanaceae) in southern India.

UNLABELLED • PREMISE OF THE STUDY In India and elsewhere, transgenic Bt eggplant (Solanum melongena) has been developed to reduce insect herbivore damage, but published studies of the potential for pollen-mediated, crop- to- wild gene flow are scant. This information is useful for risk assessments as well as in situ conservation strategies for wild germplasm.• METHODS In 2010-2014, we surveyed 23 populations of wild/weedy eggplant (Solanum insanum; known as wild brinjal), carried out hand-pollination experiments, and observed pollinators to assess the potential for crop- to- wild gene flow in southern India.• KEY RESULTS Wild brinjal is a spiny, low-growing perennial commonly found in disturbed sites such as roadsides, wastelands, and sparsely vegetated areas near villages and agricultural fields. Fourteen of the 23 wild populations in our study occurred within 0.5 km of cultivated brinjal and at least nine flowered in synchrony with the crop. Hand crosses between wild and cultivated brinjal resulted in seed set and viable F1 progeny. Wild brinjal flowers that were bagged to exclude pollinators did not set fruit, and fruit set from manual self-pollination was low. The exserted stigmas of wild brinjal are likely to promote outcrossing. The most effective pollinators appeared to be bees (Amegilla, Xylocopa, Nomia, and Heterotrigona spp.), which also were observed foraging for pollen on crop brinjal.• CONCLUSION Our findings suggest that hybridization is possible between cultivated and wild brinjal in southern India. Thus, as part of the risk assessment process, we assume that transgenes from the crop could spread to wild brinjal populations that occur nearby.

Somatic hybrids between Solanum melongena and S. sisymbrifolium, as a useful source of resistance against bacterial and fungal wilts

Abstract By using electrofusion of mesophyll protoplasts, 4 clones of somatic hybrids were produced between Solanum melongena cv. Pusa purple cluster and Solanum sisymbrifolium, a sexually incompatible wild relative resistant to bacterial ( Ralstonia solanacearum ) and fungal ( Verticillium dahliae ) wilts. All hybrids were morphologically homogeneous, normal and intermediate between the parents. They set flowers and produced fruits with empty seeds. Their hybrid status was confirmed by examining isoenzymes and random amplified polymorphism DNA analysis. Chloroplast-specific-cleaved amplified polymorphism sequence markers provided evidence that the 4 somatic hybrids possessed chloroplast genome from S. sisymbrifolium . Flow cytometry and chromosome countings revealed that all selected hybrids were tetraploids (2n=4x=48 chromosomes). The parental origin of the hybrid chromosomes was determined by using the techniques of genomic in situ hybridisation. Each hybrid contained one complete set of chromosomes from both parents. Neither spatial separation and recombinant chromosomes nor translocation could be demonstrated during mitotic metaphase. In vitro tests for resistance, using suspensions of two strains of R. solanacearum (race 1 and race 3) and the filtrate of culture medium of one strain of V. dahliae revealed that S. melongena was highly susceptible, whereas S. sisymbrifolium had variable levels of resistance. All somatic hybrids tested showed good levels of resistance, intermediate between those of the parents.

Genetic diversity and population structure of wild/weedy eggplant (Solanum insanum, Solanaceae) in southern India: Implications for conservation

UNLABELLED • PREMISE OF THE STUDY Crop wild relatives represent important genetic resources for crop improvement and the preservation of native biodiversity. Eggplant (Solanum melongena), known as brinjal in India, ranks high among crops whose wild gene pools are underrepresented in ex situ collections and warrant urgent conservation. Knowledge of outcrossing rates and patterns of genetic variation among wild populations can aid in designing strategies for both in situ and ex situ preservation.• METHODS We used 14 microsatellite (simple sequence repeat) markers to examine genetic diversity, population structure, and outcrossing in 10 natural populations of wild/weedy eggplant (S. insanum = S. melongena var. insanum) and three cultivated populations in southern India.• KEY RESULTS Multilocus FST analyses revealed strong differentiation among populations and significant isolation by distance. Bayesian model-based clustering, principal coordinate analysis, and hierarchical cluster analysis grouped the wild/weedy populations into three major clusters, largely according to their geographic origin. The three crop populations were similar to each other and grouped with two wild/weedy populations that occurred nearby. Outcrossing rates among the wild/weedy populations ranged from 5-33%, indicating a variable mixed-mating system.• CONCLUSION Geographic isolation has played a significant role in shaping the contemporary patterns of genetic differentiation among these populations, many of which represent excellent candidates for in situ conservation. In two cases, close genetic affinity between cultivars and nearby wild/weedy populations suggests that gene flow has occurred between them. To our knowledge, this is the first study investigating population-level patterns of genetic diversity in wild relatives of eggplant.

Medieval iconography of watermelons in Mediterranean Europe.

BACKGROUND AND AIMS The watermelon, Citrullus lanatus (Cucurbitaceae), is an important fruit vegetable in the warmer regions of the world. Watermelons were illustrated in Mediterranean Antiquity, but not as frequently as some other cucurbits. Little is known concerning the watermelons of Mediterranean Europe during medieval times. With the objective of obtaining an improved understanding of watermelon history and diversity in this region, medieval drawings purportedly of watermelons were collected, examined and compared for originality, detail and accuracy. FINDINGS The oldest manuscript found that contains an accurate, informative image of watermelon is the Tractatus de herbis, British Library ms. Egerton 747, which was produced in southern Italy, around the year 1300. A dozen more original illustrations were found, most of them from Italy, produced during the ensuing two centuries that can be positively identified as watermelon. In most herbal-type manuscripts, the foliage is depicted realistically, the plants shown as having long internodes, alternate leaves with pinnatifid leaf laminae, and the fruits are small, round and striped. The manuscript that contains the most detailed and accurate image of watermelon is the Carrara Herbal, British Library ms. Egerton 2020. In the agriculture-based manuscripts, the foliage, if depicted, is not accurate, but variation in the size, shape and coloration of the fruits is evident. Both red-flesh and white-flesh watermelons are illustrated, corresponding to the typical sweet dessert watermelons so common today and the insipid citron watermelons, respectively. The variation in watermelon fruit size, shape and coloration depicted in the illustrations indicates that at least six cultivars of watermelon are represented, three of which probably had red, sweet flesh and three of which appear to have been citrons. Evidently, citron watermelons were more common in Mediterranean Europe in the past than they are today.

Eggplant Resistance to the Ralstonia solanacearum Species Complex Involves Both Broad-Spectrum and Strain-Specific Quantitative Trait Loci

Bacterial wilt (BW) is a major disease of solanaceous crops caused by the Ralstonia solanacearum species complex (RSSC). Strains are grouped into five phylotypes (I, IIA, IIB, III, and IV). Varietal resistance is the most sustainable strategy for managing BW. Nevertheless, breeding to improve cultivar resistance has been limited by the pathogen’s extensive genetic diversity. Identifying the genetic bases of specific and non-specific resistance is a prerequisite to breed improvement. A major gene (ERs1) was previously mapped in eggplant (Solanum melongena L.) using an intraspecific population of recombinant inbred lines derived from the cross of susceptible MM738 (S) × resistant AG91-25 (R). ERs1 was originally found to control three strains from phylotype I, while being totally ineffective against a virulent strain from the same phylotype. We tested this population against four additional RSSC strains, representing phylotypes I, IIA, IIB, and III in order to clarify the action spectrum of ERs1. We recorded wilting symptoms and bacterial stem colonization under controlled artificial inoculation. We constructed a high-density genetic map of the population using single nucleotide polymorphisms (SNPs) developed from genotyping-by-sequencing and added 168 molecular markers [amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), and sequence-related amplified polymorphisms (SRAPs)] developed previously. The new linkage map based on a total of 1,035 markers was anchored on eggplant, tomato, and potato genomes. Quantitative trait locus (QTL) mapping for resistance against a total of eight RSSC strains resulted in the detection of one major phylotype-specific QTL and two broad-spectrum QTLs. The major QTL, which specifically controls three phylotype I strains, was located at the bottom of chromosome 9 and corresponded to the previously identified major gene ERs1. Five candidate R-genes were underlying this QTL, with different alleles between the parents. The two other QTLs detected on chromosomes 2 and 5 were found to be associated with partial resistance to strains of phylotypes I, IIA, III and strains of phylotypes IIA and III, respectively. Markers closely linked to these three QTLs will be crucial for breeding eggplant with broad-spectrum resistance to BW. Furthermore, our study provides an important contribution to the molecular characterization of ERs1, which was initially considered to be a major resistance gene.

Genotyping by Sequencing Highlights a Polygenic Resistance to Ralstonia pseudosolanacearum in Eggplant (Solanum melongena L.)

Eggplant cultivation is limited by numerous diseases, including the devastating bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC). Within the RSSC, Ralstonia pseudosolanacearum (including phylotypes I and III) causes severe damage to all solanaceous crops, including eggplant. Therefore, the creation of cultivars resistant to R. pseudosolanacearum strains is a major goal for breeders. An intraspecific eggplant population, segregating for resistance, was created from the cross between the susceptible MM738 and the resistant EG203 lines. The population of 123 doubled haploid lines was challenged with two strains belonging to phylotypes I (PSS4) and III (R3598), which both bypass the published EBWR9 BW-resistance quantitative trait locus (QTL). Ten and three QTLs of resistance to PSS4 and to R3598, respectively, were detected and mapped. All were strongly influenced by environmental conditions. The most stable QTLs were found on chromosomes 3 and 6. Given their estimated physical position, these newly detected QTLs are putatively syntenic with BW-resistance QTLs in tomato. In particular, the QTLs’ position on chromosome 6 overlaps with that of the major broad-spectrum tomato resistance QTL Bwr-6. The present study is a first step towards understanding the complex polygenic system, which underlies the high level of BW resistance of the EG203 line.