Solanum pennellii backcross inbred lines (BILs) link small genomic bins with tomato traits

We present a resource for fine mapping of traits derived from the wild tomato species Solanum pennellii (LA0716). The population of backcross inbred lines (BILs) is composed of 446 lines derived after a few generations of backcrosses of the wild species with cultivated tomato (cultivar M82; LA3475), followed by more than seven generations of self‐pollination. The BILs were genotyped using the 10K SOL‐CAP single nucleotide polymorphism (SNP) ‐Chip, and 3700 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs carry, on average, 2.7 introgressions per line, with a mean introgression length of 11.7 Mbp. Whereas the classic 76 introgression lines (ILs) partitioned the genome into 106 mapping bins, the BILs generated 633 bins, thereby enhancing the mapping resolution of traits derived from the wild species. We demonstrate the power of the BILs for rapid fine mapping of simple and complex traits derived from the wild tomato species.     

A farnesyl pyrophosphate synthase gene expressed in pollen functions in S‐RNase‐independent unilateral incompatibility

Multiple independent and overlapping pollen rejection pathways contribute to unilateral interspecific incompatibility (UI). In crosses between tomato species, pollen rejection usually occurs when the female parent is self‐incompatible (SI) and the male parent self‐compatible (SC) (the ‘SI × SC rule’). Additional, as yet unknown, UI mechanisms are independent of self‐incompatibility and contribute to UI between SC species or populations. We identified a major quantitative trait locus on chromosome 10 (ui10.1) which affects pollen‐side UI responses in crosses between cultivated tomato, Solanum lycopersicum, and Solanum pennelliiLA0716, both of which are SC and lack S‐RNase, the pistil determinant of S‐specificity in Solanaceae. Here we show that ui10.1 is a farnesyl pyrophosphate synthase gene (FPS2) expressed in pollen. Expression is about 18‐fold higher in pollen of S. pennellii than in S. lycopersicum. Pollen with the hypomorphic S. lycopersicum allele is selectively eliminated on pistils of the F₁ hybrid, leading to transmission ratio distortion in the F₂ progeny. CRISPR/Cas9‐generated knockout mutants (fps2) in S. pennelliiLA0716 are self‐sterile due to pollen rejection, but mutant pollen is fully functional on pistils of S. lycopersicum. F₂ progeny of S. lycopersicum × S. pennellii (fps2) show reversed transmission ratio distortion due to selective elimination of pollen bearing the knockout allele. Overexpression of FPS2 in S. lycopersicum pollen rescues the pollen elimination phenotype. FPS2‐based pollen selectivity does not involve S‐RNase and has not been previously linked to UI. Our results point to an entirely new mechanism of interspecific pollen rejection in plants.     

HT proteins contribute to S‐RNase‐independent pollen rejection in Solanum

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self‐incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S‐RNase and HT protein function in a pistil‐side IRB that causes rejection of pollen from self‐compatible (SC) red/orange‐fruited species in the tomato clade. However, S‐RNase‐independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S‐RNase‐independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S‐RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S‐RNase, and then used RNAi to test whether they also function in S‐RNase‐independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S‐RNase‐dependent and S‐RNase‐independent pollen rejection. The results support the view that overall compatibility results from multiple pollen–pistil interactions with additive effects.     

Detection of major loci associated with the variation of 18 important agronomic traits between Solanum pimpinellifolium and cultivated tomatoes

Wild species can be used to improve various agronomic traits in cultivars; however, a limited understanding of the genetic basis underlying the morphological differences between wild and cultivated species hinders the integration of beneficial traits from wild species. In the present study, we generated and sequenced recombinant inbred lines (RILs, 201 F₁₀ lines) derived from a cross between Solanum pimpinellifolium and Solanum lycopersicum tomatoes. Based on a high‐resolution recombination bin map to uncover major loci determining the phenotypic variance between wild and cultivated tomatoes, 104 significantly associated loci were identified for 18 agronomic traits. On average, these loci explained ~39% of the phenotypic variance of the RILs. We further generated near‐isogenic lines (NILs) for four identified loci, and the lines exhibited significant differences for the associated traits. We found that two loci could improve the flower number and inflorescence architecture in the cultivar following introgression of the wild‐species alleles. These findings allowed us to construct a trait–locus network to help explain the correlations among different traits based on the pleiotropic or linked loci. Our results provide insights into the morphological changes between wild and cultivated tomatoes, and will help to identify key genes governing important agronomic traits for the molecular selection of elite tomato varieties.     

Introgression lines of Solanum sitiens,a wild nightshade of the Atacama Desert,in the genome of cultivated tomato

The wild tomato relative Solanum sitiens is a xerophyte endemic to the Atacama Desert of Chile and a potential source of genes for tolerance to drought, salinity and low‐temperature stresses. However, until recently, strong breeding barriers prevented its hybridization and introgression with cultivated tomato, Solanum lycopersicum L. We overcame these barriers using embryo rescue, bridging lines and allopolyploid hybrids, and synthesized a library of introgression lines (ILs) that captures the genome of S. sitiens in the background of cultivated tomato. The IL library consists of 56 overlapping introgressions that together represent about 93% of the S. sitiens genome: 65% in homozygous and 28% in heterozygous (segregating) ILs. The breakpoints of each segment and the gaps in genome coverage were mapped by single nucleotide polymorphism (SNP) genotyping using the SolCAP SNP array. Marker‐assisted selection was used to backcross selected introgressions into tomato, to recover a uniform genetic background, to isolate recombinant sub‐lines with shorter introgressions and to select homozygous genotypes. Each IL contains a single S. sitiens chromosome segment, defined by markers, in the genetic background of cv. NC 84173, a fresh market inbred line. Large differences were observed between the lines for both qualitative and quantitative morphological traits, suggesting that the ILs contain highly divergent allelic variation. Several loci contributing to unilateral incompatibility or hybrid necrosis were mapped with the lines. This IL population will facilitate studies of the S. sitiens genome and expands the range of genetic variation available for tomato breeding and research.     

Salt tolerance in <Emphasis Type="Italic">Solanum pennellii</Emphasis>: antioxidant response and related QTL

Background  

Excessive soil salinity is an important problem for agriculture, however, salt tolerance is a complex trait that is not easily bred into plants. Exposure of cultivated tomato to salt stress has been reported to result in increased antioxidant content and activity. Salt tolerance of the related wild species, Solanum pennellii, has also been associated with similar changes in antioxidants. In this work, S. lycopersicum M82, S. pennellii LA716 and a S. pennellii introgression line (IL) population were evaluated for growth and their levels of antioxidant activity (total water-soluble antioxidant activity), major antioxidant compounds (phenolic and flavonoid contents) and antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase and peroxidase) under both control and salt stress (150 mM NaCl) conditions. These data were then used to identify quantitative trait loci (QTL) responsible for controlling the antioxidant parameters under both stress and nonstress conditions.     

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB‐LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB‐LRR (nucleotide binding‐site leucine‐rich repeat) gene‐targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB‐LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB‐LRRs and can be accessed through a genome browser that we provide. We compared these NB‐LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB‐LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co‐segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi‐ber2) and S. ruiz‐ceballosii (Rpi‐rzc1), we were able to apply RenSeq successfully to identify markers that co‐segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy‐to‐adapt Galaxy pipelines.     

Ptr1 evolved convergently with RPS2 and Mr5 to mediate recognition of AvrRpt2 in diverse solanaceous species

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide‐binding leucine‐rich repeat protein (NLR)‐encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA‐Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in Nicotiana benthamiana and potato, and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared with Mr5 and RPS2, and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.     

Oviposition dynamics and larval development of Helicoverpa armigera on a highly preferred unsuitable host plant,Solanum viarum

Oogenesis and oviposition dynamics of female moths of tomato fruitworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae), were compared in a monoculture of tomato (Solanum lycopersicum L.) and in mixtures with a highly preferred but unsuitable host plant, Solanum viarum Dunal. Female H. armigera provided with S. viarum laid many more eggs than those provided with tomato, both in choice and no‐choice experiments. Gravid female moths did not display any preference for oviposition neither between young or old plants of S. viarum or tomato, nor between young and old leaves of S. viarum. Larval mortality and larval developmental period significantly increased, with subsequent reduction in pupal weight, when H. armigera larvae fed on S. viarum leaves compared to a meridic diet. Oviposition of female moths significantly increased in tomato in the presence of S. viarum volatiles. However, in screen cages with an increasing percentage of S. viarum as a trap crop, the number of eggs laid on tomato as the main crop was the same. The results of this study are discussed in light of the possibility of using S. viarum as a ‘dead‐end’ trap crop.     

Genome sequence of M6, a diploid inbred clone of the high‐glycoalkaloid‐producing tuber‐bearing potato species Solanum chacoense,reveals residual heterozygosity

Cultivated potato (Solanum tuberosum L.) is a highly heterozygous autotetraploid that presents challenges in genome analyses and breeding. Wild potato species serve as a resource for the introgression of important agronomic traits into cultivated potato. One key species is Solanum chacoense and the diploid, inbred clone M6, which is self‐compatible and has desirable tuber market quality and disease resistance traits. Sequencing and assembly of the genome of the M6 clone of S. chacoense generated an assembly of 825 767 562 bp in 8260 scaffolds with an N50 scaffold size of 713 602 bp. Pseudomolecule construction anchored 508 Mb of the genome assembly into 12 chromosomes. Genome annotation yielded 49 124 high‐confidence gene models representing 37 740 genes. Comparative analyses of the M6 genome with six other Solanaceae species revealed a core set of 158 367 Solanaceae genes and 1897 genes unique to three potato species. Analysis of single nucleotide polymorphisms across the M6 genome revealed enhanced residual heterozygosity on chromosomes 4, 8 and 9 relative to the other chromosomes. Access to the M6 genome provides a resource for identification of key genes for important agronomic traits and aids in genome‐enabled development of inbred diploid potatoes with the potential to accelerate potato breeding.     

A TILLING approach to generate broad‐spectrum resistance to potyviruses in tomato is hampered by eIF4E gene redundancy

Genetic resistance to pathogens is important for sustainable maintenance of crop yields. Recent biotechnologies offer alternative approaches to generate resistant plants by compensating for the lack of natural resistance. Tomato (Solanum lycopersicum) and related species offer a model in which natural and TILLING‐induced potyvirus resistance alleles may be compared. For resistance based on translation initiation factor eIF4E1, we confirm that the natural allele Sh–eIF4E1^PI24–pot1, isolated from the wild tomato species Solanum habrochaites, is associated with a wide spectrum of resistance to both potato virus Y and tobacco etch virus isolates. In contrast, a null allele of the same gene, isolated through a TILLING strategy in cultivated tomato S. lycopersicum, is associated with a much narrower resistance spectrum. Introgressing the null allele into S. habrochaites did not extend its resistance spectrum, indicating that the genetic background is not responsible for the broad resistance. Instead, the different types of eIF4E1 mutations affect the levels of eIF4E2 differently, suggesting that eIF4E2 is also involved in potyvirus resistance. Indeed, combining two null mutations affecting eIF4E1 and eIF4E2 re‐establishes a wide resistance spectrum in cultivated tomato, but to the detriment of plant development. These results highlight redundancy effects within the eIF4E gene family, where regulation of expression alters susceptibility or resistance to potyviruses. For crop improvement, using loss‐of‐function alleles to generate resistance may be counter‐productive if they narrow the resistance spectrum and limit growth. It may be more effective to use alleles encoding functional variants similar to those found in natural diversity.     

Non‐GMO potato lines,synthesizing increased amylose and resistant starch,are mainly deficient in isoamylase debranching enzyme

Solanum tuberosum potato lines with high amylose content were generated by crossing with the wild potato species Solanum sandemanii followed by repeated backcrossing to Solanum tuberosum lines. The trait, termed increased amylose (IAm), was recessive and present after three generations of backcrossing into S. tuberosum lines (6.25% S. sandemanii genes). The tubers of these lines were small, elongated and irregular with small and misshaped starch granules and high sugar content. Additional backcrossing resulted in less irregular tuber morphology, increased starch content (4.3%–9.5%) and increased amylose content (29%–37.9%) but indifferent sugar content. The amylose in the IAm starch granules was mainly located in peripheral spots, and large cavities were found in the granules. Starch pasting was suppressed, and the digestion‐resistant starch (RS) content was increased. Comprehensive microarray polymer profiling (CoMPP) analysis revealed specific alterations of major pectic and glycoprotein cell wall components. This complex phenotype led us to search for candidate IAm genes exploiting its recessive trait. Hence, we sequenced genomic DNA of a pool of IAm lines, identified SNPs genome wide against the draft genome sequence of potato and searched for regions of decreased heterozygosity. Three regions, located on chromosomes 3, 7 and 10, respectively, displayed markedly less heterozygosity than average. The only credible starch metabolism‐related gene found in these regions encoded the isoamylase‐type debranching enzyme Stisa1. Decreased expression of mRNA (>500 fold) and reduced enzyme activity (virtually absent from IAm lines) supported Stisa1 as a candidate gene for IAm.     

Biocontrol of Fusarium Wilt and Growth Promotion of Tomato Plants Using Endophytic Bacteria Isolated from Solanum elaeagnifolium Stems

Seven culturable bacterial isolates, obtained from the internal stem tissues of Solanum elaeagnifolium and successfully colonizing the internal stem tissues of tomato cv. Rio Grande, were screened for their in vivo antifungal activity against Fusarium oxysporum f.sp. lycopersici (FOL) and their growth‐promoting potential on tomato plants. SV101 and SV104 isolates, assessed on pathogen‐challenged tomato plants led to a significant decrease (77–83%) in Fusarium wilt severity and vascular browning extent (76%), as compared to the inoculated and untreated control. Isolates enhanced growth parameters on pathogen‐challenged and unchallenged tomato plants. SV104 and SV101 isolates were most effective in suppressing disease and enhancing plant growth. These two isolates were identified as Bacillus sp. str. SV101 ( KU043040 ) and B. tequilensis str. SV104 ( KU976970 ). They displayed antifungal activity against FOL; pathogen growth was inhibited by 64% and an inhibition zone (11.50 and 19.75 mm) against FOL could be formed using whole cell suspensions. SV101 and SV104 extracellular metabolites also inhibited FOL growth by 20 and 55%, respectively, as compared to control. B. tequilensis str. SV104 was shown to produce protease, chitinase, pectinase, IAA and siderophores. Bacillus sp. str. SV101 displayed pectinase activity and was found to be an IAA‐producing and phosphate‐solubilizing agent. To our knowledge, this is the first study reporting on S. elaeagnifolium use as a potential source of potent biocontrol and plant growth‐promoting agents.     

Tolerance and overcompensation to infection by Phytophthora infestans in the wild perennial climber Solanum dulcamara

Studies of infection by Phytophthora infestans—the causal agent of potato late blight—in wild species can provide novel insights into plant defense responses, and indicate how wild plants might be influenced by recurrent epidemics in agricultural fields. In the present study, our aim was to investigate if different clones of Solanum dulcamara (a relative of potato) collected in the wild differ in resistance and tolerance to infection by a common European isolate of P. infestans. We performed infection experiments with six S. dulcamara genotypes (clones) both in the laboratory and in the field and measured the degree of infection and plant performance traits. In the laboratory, the six evaluated genotypes varied from resistant to susceptible, as measured by degree of infection 20 days post infection. Two of the four genotypes susceptible to infection showed a quadratic (concave downward) relationship between the degree of infection and shoot length, with maximum shoot length at intermediate values of infection. This result suggests overcompensation, that is, an increase in growth in infected individuals. The number of leaves decreased with increasing degree of infection, but at different rates in the four susceptible genotypes, indicating genetic variation for tolerance. In the field, the inoculated genotypes did not show any disease symptoms, but plant biomass at the end of the growing season was higher for inoculated plants than for controls, in‐line with the overcompensation detected in the laboratory. We conclude that in S. dulcamara there are indications of genetic variation for both resistance and tolerance to P. infestans infection. Moreover, some genotypes displayed overcompensation. Learning about plant tolerance and overcompensation to infection by pathogens can help broaden our understanding of plant defense in natural populations and help develop more sustainable plant protection strategies for economically important crop diseases.     

Next‐generation sequencing (NGS)‐based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next‐generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty‐five genes belonging to carotenoids and folate metabolism were PCR‐amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600‐bp amplicons were directly sequenced in a non‐overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128‐fold pooling. An evaluation of six different software programs (camba , crisp , gatk unified genotyper , lofreq , snver and vipr ) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.     

Identification of the carotenoid modifying gene PALE YELLOW PETAL 1 as an essential factor in xanthophyll esterification and yellow flower pigmentation in tomato (Solanum lycopersicum)

Xanthophylls, the pigments responsible for yellow to red coloration, are naturally occurring carotenoid compounds in many colored tissues of plants. These pigments are esterified within the chromoplast; however, little is known about the mechanisms underlying their accumulation in flower organs. In this study, we characterized two allelic tomato (Solanum lycopersicum L.) mutants, pale yellow petal (pyp) 1‐1 and pyp1‐2, that have reduced yellow color intensity in the petals and anthers due to loss‐of‐function mutations. Carotenoid analyses showed that the yellow flower organs of wild‐type tomato contained high levels of xanthophylls that largely consisted of neoxanthin and violaxanthin esterified with myristic and/or palmitic acids. Functional disruption of PYP1 resulted in loss of xanthophyll esters, which was associated with a reduction in the total carotenoid content and disruption of normal chromoplast development. These findings suggest that xanthophyll esterification promotes the sequestration of carotenoids in the chromoplast and that accumulation of these esters is important for normal chromoplast development. Next‐generation sequencing coupled with map‐based positional cloning identified the mutant alleles responsible for the pyp1 phenotype. PYP1 most likely encodes a carotenoid modifying protein that plays a vital role in the production of xanthophyll esters in tomato anthers and petals. Our results provide insight into the molecular mechanism underlying the production of xanthophyll esters in higher plants, thereby shedding light on a longstanding mystery.     

Identification of the New Pathogen (Stemphylium lycopersici) Causing Leaf Spot on Pepino (Solanum muricatum)

Pepino (Solanum muricatum var. pepino) plants were found affected by an extensive leaf spot caused by plant pathogenic fungi during a survey in the Cameron highlands, Pahang state, Malaysia. Symptomatic leaf samples were collected from infected pepino plants and cultivated on PDA medium, and the pathogen was isolated and purified; then, consequently, all isolates were identified as Stemphylium lycopersici on the basis of their cultural and morphological characteristics and combined sequences of the internal transcribed spacer (ITS) and glyceraldehyde‐3‐phosphate dehydrogenase (gpd) regions. A pathogenicity assay on detached leaves further confirmed that S. lycopersici causes leaf spot disease. To the best of our knowledge, this is the first report of S. lycopersici causing leaf spot on pepino in Malaysia and worldwide.     

The Ficus erecta genome aids Ceratocystis canker resistance breeding in common fig (F. carica)

Ficus erecta, a wild relative of the common fig (F. carica), is a donor of Ceratocystis canker resistance in fig breeding programmes. Interspecific hybridization followed by recurrent backcrossing is an effective method to transfer the resistance trait from wild to cultivated fig. However, this process is time consuming and labour intensive for trees, especially for gynodioecious plants such as fig. In this study, genome resources were developed for F. erecta to facilitate fig breeding programmes. The genome sequence of F. erecta was determined using single‐molecule real‐time sequencing technology. The resultant assembly spanned 331.6 Mb with 538 contigs and an N50 length of 1.9 Mb, from which 51 806 high‐confidence genes were predicted. Pseudomolecule sequences corresponding to the chromosomes of F. erecta were established with a genetic map based on single nucleotide polymorphisms from double‐digest restriction‐site‐associated DNA sequencing. Subsequent linkage analysis and whole‐genome resequencing identified a candidate gene for the Ceratocystis canker resistance trait. Genome‐wide genotyping analysis enabled the selection of female lines that possessed resistance and effective elimination of the donor genome from the progeny. The genome resources provided in this study will accelerate and enhance disease‐resistance breeding programmes in fig.