ETHQV6.3 is involved in melon climacteric fruit ripening and is encoded by a NAC domain transcription factor.

Fruit ripening is divided into climacteric and non-climacteric types depending on the presence or absence of a transient rise in respiration rate and the production of autocatalytic ethylene. Melon is ideal for the study of fruit ripening, as both climacteric and non-climacteric varieties exist. Two introgressions of the non-climacteric accession PI 161375, encompassed in the QTLs ETHQB3.5 and ETHQV6.3, into the non-climacteric 'Piel de Sapo' background are able to induce climacteric ripening independently. We report that the gene underlying ETHQV6.3 is MELO3C016540 (CmNAC-NOR), encoding a NAC (NAM, ATAF1,2, CUC2) transcription factor that is closely related to the tomato NOR (non-ripening) gene. CmNAC-NOR was functionally validated through the identification of two TILLING lines carrying non-synonymous mutations in the conserved NAC domain region. In an otherwise highly climacteric genetic background, both mutations provoked a significant delay in the onset of fruit ripening and in the biosynthesis of ethylene. The PI 161375 allele of ETHQV6.3 is similar to that of climacteric lines of the cantalupensis type and, when introgressed into the non-climacteric 'Piel de Sapo', partially restores its climacteric ripening capacity. CmNAC-NOR is expressed in fruit flesh of both climacteric and non-climacteric lines, suggesting that the causal mutation may not be acting at the transcriptional level. The use of a comparative genetic approach in a species with both climacteric and non-climacteric ripening is a powerful strategy to dissect the complex mechanisms regulating the onset of fruit ripening.

Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly.

BACKGROUND: The genome of the melon (Cucumis melo L.) double-haploid line DHL92 was recently sequenced, with 87.5 and 80.8% of the scaffold assembly anchored and oriented to the 12 linkage groups, respectively. However, insufficient marker coverage and a lack of recombination left several large, gene rich scaffolds unanchored, and some anchored scaffolds unoriented. To improve the anchoring and orientation of the melon genome assembly, we used resequencing data between the parental lines of DHL92 to develop a new set of SNP markers from unanchored scaffolds. RESULTS: A high-resolution genetic map composed of 580 SNPs was used to anchor 354.8 Mb of sequence, contained in 141 scaffolds (average size 2.5 Mb) and corresponding to 98.2% of the scaffold assembly, to the 12 melon chromosomes. Over 325.4 Mb (90%) of the assembly was oriented. The genetic map revealed regions of segregation distortion favoring SC alleles as well as recombination suppression regions coinciding with putative centromere, 45S, and 5S rDNA sites. New chromosome-scale pseudomolecules were created by incorporating to the previous v3.5 version an additional 38.3 Mb of anchored sequence representing 1,837 predicted genes contained in 55 scaffolds. Using fluorescent in situ hybridization (FISH) with BACs that produced chromosome-specific signals, melon chromosomes that correspond to the twelve linkage groups were identified, and a standardized karyotype of melon inbred line T111 was developed. CONCLUSIONS: By utilizing resequencing data and targeted SNP selection combined with a large F2 mapping population, we significantly improved the quantity of anchored and oriented melon scaffold genome assembly. Using genome information combined with FISH mapping provided the first cytogenetic map of an inodorus melon type. With these results it was possible to make inferences on melon chromosome structure by relating zones of recombination suppression to centromeres and 45S and 5S heterochromatic regions. This study represents the first steps towards the integration of the high-resolution genetic and cytogenetic maps with the genomic sequence in melon that will provide more information on genome organization and allow for the improvement of the melon genome draft sequence.

Combined use of genetic and genomics resources to understand virus resistance and fruit quality traits in melon.

The availability of the genome sequence of many crop species during the past few years has opened a new era in plant biology, allowing for the performance of massive genomic studies in plant species other than the classical models Arabidopsis and rice. One of these crop species is melon (Cucumis melo), a cucurbit of high economic value that has become an interesting model for the study of biological processes such as fruit ripening, sex determination and phloem transport. The recent availability of the melon genome sequence, together with a number of genetic and genomic resources, provides powerful tools that can be used to assist in the main melon breeding targets, namely disease resistance and fruit quality. In this review, we will describe recent data obtained combining the use of a melon near isogenic line (NIL) population and genomic resources to gain insight into agronomically important traits as fruit ripening, resistance to Cucumber Mosaic virus (CMV) and the accumulation of sugars in fruits.

High-throughput methods to identify male Cannabis sativa using various genotyping methods.

BACKGROUND: Cannabis sativa is a primarily dioecious angiosperm that exhibits sexual developmental plasticity. Developmental genes for staminate male flowers have yet to be elucidated; however, there are regions of male-associated DNA from Cannabis (MADC) that correlate with the formation of pollen producing staminate flowers. MADC2 is an example of a PCR-based genetic marker that has been shown to produce a 390-bp amplicon that correlates with the expression of male phenotypes. We demonstrate applications of a cost-effective high-throughput male genotyping assay and other genotyping applications of male identification in Cannabis sativa. METHODS: In this study, we assessed data from 8200 leaf samples analyzed for real-time quantitative polymerase chain reaction (qPCR) detection of MADC2 in a commercial testing application offered through Steep Hill Laboratories. Through validation, collaborative research projects, and follow-up retest analysis, we observed a > 98.5% accuracy of detection of MADC2 by qPCR. We also carried out assay development for high-resolution melting analysis (HRM), loop-mediated isothermal amplification (LAMP), and TwistDx recombinase amplification (RPA) assays using MADC2 for male identification. RESULTS: We demonstrate a robust high-throughput duplex TaqMan qPCR assay for identification of male-specific genomic signatures using a novel MADC2 qPCR probe. The qPCR cycle quotient (Cq) value representative of MADC2 detection in 3156 males and the detection of tissue control cannabinoid synthesis for 8200 samples and the absence of MADC2 detection in 5047 non-males demonstrate a robust high-throughput real-time genotyping assay for Cannabis. Furthermore, we also demonstrated the viability of using nearby regions to MADC2 with novel primers as alternative assays. Finally, we also show proof of concept of several additional commercially viable sex determination methodologies for Cannabis sativa. DISCUSSION: In industrial applications, males are desirable for their more rapid growth and higher quality fiber quality, as well as their ability to pollinate female plants and produce grain. In medicinal applications, female cultivars are more desirable for their ability to produce large amounts of secondary metabolites, specifically the cannabinoids, terpenes, and flavonoids that have various medicinal and recreational properties. In previous studies, traditional PCR and non-high-throughput methods have been reported for the detection of male cannabis, and in our study, we present multiple methodologies that can be carried out in high-throughput commercial cannabis testing. CONCLUSION: With these markers developed for high-throughput testing assays, the Cannabis industry will be able to easily screen and select for the desired sex of a given cultivar depending on the application.

Fruit Morphology and Ripening-Related QTLs in a Newly Developed Introgression Line Collection of the Elite Varieties 'Védrantais' and 'Piel de Sapo'.

Melon is an economically important crop with widely diverse fruit morphology and ripening characteristics. Its diploid sequenced genome and multiple genomic tools make this species suitable to study the genetic architecture of fruit traits. With the development of this introgression line population of the elite varieties 'Piel de Sapo' and 'Védrantais', we present a powerful tool to study fruit morphology and ripening traits that can also facilitate characterization or pyramidation of QTLs in inodorous melon types. The population consists of 36 lines covering almost 98% of the melon genome, with an average of three introgressions per chromosome and segregating for multiple fruit traits: morphology, ripening and quality. High variability in fruit morphology was found within the population, with 24 QTLs affecting six different traits, confirming previously reported QTLs and two newly detected QTLs, FLQW5.1 and FWQW7.1. We detected 20 QTLs affecting fruit ripening traits, six of them reported for the first time, two affecting the timing of yellowing of the rind (EYELLQW1.1 and EYELLQW8.1) and four at the end of chromosome 8 affecting aroma, abscission and harvest date (EAROQW8.3, EALFQW8.3, ABSQW8.3 and HARQW8.3). We also confirmed the location of several QTLs, such as fruit-quality-related QTLs affecting rind and flesh appearance and flesh firmness.

A gene encoding an abscisic acid biosynthetic enzyme (LsNCED4) collocates with the high temperature germination locus Htg6.1 in lettuce (Lactuca sp.).

Thermoinhibition, or failure of seeds to germinate when imbibed at warm temperatures, can be a significant problem in lettuce (Lactuca sativa L.) production. The reliability of stand establishment would be improved by increasing the ability of lettuce seeds to germinate at high temperatures. Genes encoding germination- or dormancy-related proteins were mapped in a recombinant inbred line population derived from a cross between L. sativa cv. Salinas and L. serriola accession UC96US23. This revealed several candidate genes that are located in the genomic regions containing quantitative trait loci (QTLs) associated with temperature and light requirements for germination. In particular, LsNCED4, a temperature-regulated gene in the biosynthetic pathway for abscisic acid (ABA), a germination inhibitor, mapped to the center of a previously detected QTL for high temperature germination (Htg6.1) from UC96US23. Three sets of sister BC(3)S(2) near-isogenic lines (NILs) that were homozygous for the UC96US23 allele of LsNCED4 at Htg6.1 were developed by backcrossing to cv. Salinas and marker-assisted selection followed by selfing. The maximum temperature for germination of NIL seed lots with the UC96US23 allele at LsNCED4 was increased by 2-3°C when compared with sister NIL seed lots lacking the introgression. In addition, the expression of LsNCED4 was two- to threefold lower in the former NIL lines as compared to expression in the latter. Together, these data strongly implicate LsNCED4 as the candidate gene responsible for the Htg6.1 phenotype and indicate that decreased ABA biosynthesis at high imbibition temperatures is a major factor responsible for the increased germination thermotolerance of UC96US23 seeds.

A novel introgression line collection to unravel the genetics of climacteric ripening and fruit quality in melon.

Introgression lines are valuable germplasm for scientists and breeders, since they ease genetic studies such as QTL interactions and positional cloning as well as the introduction of favorable alleles into elite varieties. We developed a novel introgression line collection in melon using two commercial European varieties with different ripening behavior, the climacteric cantalupensis 'Védrantais' as recurrent parent and the non-climacteric inodorus 'Piel de Sapo' as donor parent. The collection contains 34 introgression lines, covering 99% of the donor genome. The mean introgression size is 18.16 Mb and ~ 3 lines were obtained per chromosome, on average. The high segregation of these lines for multiple fruit quality traits allowed us to identify 27 QTLs that modified sugar content, altered fruit morphology or were involved in climacteric ripening. In addition, we confirmed the genomic location of five major genes previously described, which control mainly fruit appearance, such as mottled rind and external color. Most of the QTLs had been reported before in other populations sharing parental lines, while three QTLs (EAROQP11.3, ECDQP11.2 and FIRQP4.1) were newly detected in our work. These introgression lines would be useful to perform additional genetic studies, as fine mapping and gene pyramiding, especially for important complex traits such as fruit weight and climacteric ripening.

Genetic dissection of climacteric fruit ripening in a melon population segregating for ripening behavior.

Melon is as an alternative model to understand fruit ripening due to the coexistence of climacteric and non-climacteric varieties within the same species, allowing the study of the processes that regulate this complex trait with genetic approaches. We phenotyped a population of recombinant inbred lines (RILs), obtained by crossing a climacteric (Védrantais, cantalupensis type) and a non-climcteric variety (Piel de Sapo T111, inodorus type), for traits related to climacteric maturation and ethylene production. Individuals in the RIL population exhibited various combinations of phenotypes that differed in the amount of ethylene produced, the early onset of ethylene production, and other phenotypes associated with ripening. We characterized a major QTL on chromosome 8, ETHQV8.1, which is sufficient to activate climacteric ripening, and other minor QTLs that may modulate the climacteric response. The ETHQV8.1 allele was validated by using two reciprocal introgression line populations generated by crossing Védrantais and Piel de Sapo and analyzing the ETHQV8.1 region in each of the genetic backgrounds. A Genome-wide association study (GWAS) using 211 accessions of the ssp. melo further identified two regions on chromosome 8 associated with the production of aromas, one of these regions overlapping with the 154.1 kb interval containing ETHQV8.1. The ETHQV8.1 region contains several candidate genes that may be related to fruit ripening. This work sheds light into the regulation mechanisms of a complex trait such as fruit ripening.

A comprehensive genome variation map of melon identifies multiple domestication events and loci influencing agronomic traits.

Melon is an economically important fruit crop that has been cultivated for thousands of years; however, the genetic basis and history of its domestication still remain largely unknown. Here we report a comprehensive map of the genomic variation in melon derived from the resequencing of 1,175 accessions, which represent the global diversity of the species. Our results suggest that three independent domestication events occurred in melon, two in India and one in Africa. We detected two independent sets of domestication sweeps, resulting in diverse characteristics of the two subspecies melo and agrestis during melon breeding. Genome-wide association studies for 16 agronomic traits identified 208 loci significantly associated with fruit mass, quality and morphological characters. This study sheds light on the domestication history of melon and provides a valuable resource for genomics-assisted breeding of this important crop.

Genome encode analyses reveal the basis of convergent evolution of fleshy fruit ripening.

Fleshy fruits using ethylene to regulate ripening have developed multiple times in the history of angiosperms, presenting a clear case of convergent evolution whose molecular basis remains largely unknown. Analysis of the fruitENCODE data consisting of 361 transcriptome, 71 accessible chromatin, 147 histone and 45 DNA methylation profiles reveals three types of transcriptional feedback circuits controlling ethylene-dependent fruit ripening. These circuits are evolved from senescence or floral organ identity pathways in the ancestral angiosperms either by neofunctionalisation or repurposing pre-existing genes. The epigenome, H3K27me3 in particular, has played a conserved role in restricting ripening genes and their orthologues in dry and ethylene-independent fleshy fruits. Our findings suggest that evolution of ripening is constrained by limited hormone molecules and genetic and epigenetic materials, and whole-genome duplications have provided opportunities for plants to successfully circumvent these limitations.

An improved assembly and annotation of the melon (Cucumis melo L.) reference genome.

We report an improved assembly (v3.6.1) of the melon (Cucumis melo L.) genome and a new genome annotation (v4.0). The optical mapping approach allowed correcting the order and the orientation of 21 previous scaffolds and permitted to correctly define the gap-size extension along the 12 pseudomolecules. A new comprehensive annotation was also built in order to update the previous annotation v3.5.1, released more than six years ago. Using an integrative annotation pipeline, based on exhaustive RNA-Seq collections and ad-hoc transposable element annotation, we identified 29,980 protein-coding loci. Compared to the previous version, the v4.0 annotation improved gene models in terms of completeness of gene structure, UTR regions definition, intron-exon junctions and reduction of fragmented genes. More than 8,000 new genes were identified, one third of them being well supported by RNA-Seq data. To make all the new resources easily exploitable and completely available for the scientific community, a redesigned Melonomics genomic platform was released at http://melonomics.net . The resources produced in this work considerably increase the reliability of the melon genome assembly and resolution of the gene models paving the way for further studies in melon and related species.

QTL Analyses in Multiple Populations Employed for the Fine Mapping and Identification of Candidate Genes at a Locus Affecting Sugar Accumulation in Melon (Cucumis melo L.).

Sugar content is the major determinant of both fruit quality and consumer acceptance in melon (Cucumis melo L), and is a primary target for crop improvement. Near-isogenic lines (NILs) derived from the intraspecific cross between a "Piel de Sapo" (PS) type and the exotic cultivar "Songwhan Charmi" (SC), and several populations generated from the cross of PS × Ames 24294 ("Trigonus"), a wild melon, were used to identify QTL related to sugar and organic acid composition. Seventy-eight QTL were detected across several locations and different years, with three important clusters related to sugar content located on chromosomes 4, 5, and 7. Two PS × SC NILs (SC5-1 and SC5-2) sharing a common genomic interval of 1.7 Mb at the top of chromosome 5 contained QTL reducing soluble solids content (SSC) and sucrose content by an average of 29 and 68%, respectively. This cluster collocated with QTL affecting sugar content identified in other studies in lines developed from the PS × SC cross and supported the presence of a stable consensus locus involved in sugar accumulation that we named SUCQSC5.1. QTL reducing soluble solids and sucrose content identified in the "Trigonus" mapping populations, as well as QTL identified in previous studies from other ssp. agrestis sources, collocated with SUCQSC5.1, suggesting that they may be allelic and implying a role in domestication. In subNILs derived from the PS × SC5-1 cross, SUCQSC5.1 reduced SSC and sucrose content by an average of 18 and 34%, respectively, and was fine-mapped to a 56.1 kb interval containing four genes. Expression analysis of the candidate genes in mature fruit showed differences between the subNILs with PS alleles that were "high" sugar and SC alleles of "low" sugar phenotypes for MELO3C014519, encoding a putative BEL1-like homeodomain protein. Sequence differences in the gene predicted to affect protein function were restricted to SC and other ssp. agrestis cultivar groups. These results provide the basis for further investigation of genes affecting sugar accumulation in melon.

Genetic variation for lettuce seed thermoinhibition is associated with temperature-sensitive expression of abscisic Acid, gibberellin, and ethylene biosynthesis, metabolism, and response genes.

Lettuce (Lactuca sativa 'Salinas') seeds fail to germinate when imbibed at temperatures above 25 degrees C to 30 degrees C (termed thermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37 degrees C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associated with these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.

The self-incompatibility locus (S) and quantitative trait loci for self-pollination and seed dormancy in sunflower.

Wild populations of common sunflower (Helianthus annuus L.) are self-incompatible and have deep seed dormancy, whereas modern cultivars, inbreds, and hybrids are self-compatible and partially-to-strongly self-pollinated, and have shallow seed dormancy. Self-pollination (SP) and seed dormancy are genetically complex traits, the number of self-compatibility (S) loci has been disputed, and none of the putative S loci have been genetically mapped in sunflower. We genetically mapped quantitative trait loci (QTL) for self-incompatibility (SI), SP, and seed dormancy in a backcross population produced from a cross between an elite, self-pollinated, nondormant inbred line (NMS373) and a wild, self-incompatible, dormant population (ANN1811). A population consisting of 212 BC(1) progeny was subsequently produced by backcrossing a single hybrid individual to NMS373. BC(1) progeny produced 0-838 seeds per primary capitula when naturally selfed and 0-518 seeds per secondary capitula when manually selfed and segregated for a single S locus. The S locus mapped to linkage group 17 and was tightly linked to a cluster of previously identified QTL for several domestication and postdomestication traits. Two synergistically interacting QTL were identified for SP among self-compatible (ss) BC(1) progeny (R(2)=34.6%). NMS373 homozygotes produced 271.5 more seeds per secondary capitulum than heterozygotes. Germination percentages of seeds after-ripened for 4 weeks ranged from 0% to 100% among self-compatible BC(1)S(1) families. Three QTL for seed dormancy were identified (R(2)=38.3%). QTL effects were in the predicted direction (wild alleles decreased self-pollination and seed germination). The present analysis differentiated between loci governing SI and SP and identified DNA markers for bypassing SI and seed dormancy in elite x wild crosses through marker-assisted selection.

Quantitative trait loci associated with seed and seedling traits in Lactuca.

Seed and seedling traits related to germination and stand establishment are important in the production of cultivated lettuce (Lactuca sativa L.). Six seed and seedling traits segregating in a L. sativa cv. Salinas x L. serriola recombinant inbred line population consisting of 103 F8 families revealed a total of 17 significant quantitative trait loci (QTL) resulting from three seed production environments. Significant QTL were identified for germination in darkness, germination at 25 and 35 degrees C, median maximum temperature of germination, hypocotyl length at 72 h post-imbibition, and plant (seedling) quality. Some QTL for germination and early seedling growth characteristics were co-located, suggestive of pleiotropic loci regulating these traits. A single QTL (Htg6.1) described 25 and 23% of the total phenotypic variation for high temperature germination in California- and Netherlands-grown populations, respectively, and was significant between 33 and 37 degrees C. Additionally, Htg6.1 showed significant epistatic interactions with other Htg QTL and a consistent effect across all the three seed production environments. L. serriola alleles increased germination at these QTL. The estimate of narrow-sense heritability (h2) of Htg6.1 was 0.84, indicating potential for L. serriola as a source of germination thermotolerance for lettuce introgression programs.