Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.

Population genomic analyses from low-coverage RAD-Seq data: a case study on the non-model cucurbit bottle gourd.

Restriction site-associated DNA sequencing (RAD-Seq), a next-generation sequencing-based genome 'complexity reduction' protocol, has been useful in population genomics in species with a reference genome. However, the application of this protocol to natural populations of genomically underinvestigated species, particularly under low-to-medium sequencing depth, has not been well justified. In this study, a Bayesian method was developed for calling genotypes from an F2 population of bottle gourd [Lagenaria siceraria (Mol.) Standl.] to construct a high-density genetic map. Low-depth genome shotgun sequencing allowed the assembly of scaffolds/contigs comprising approximately 50% of the estimated genome, of which 922 were anchored for identifying syntenic regions between species. RAD-Seq genotyping of a natural population comprising 80 accessions identified 3226 single nuclear polymorphisms (SNPs), based on which two sub-gene pools were suggested for association with fruit shape. The two sub-gene pools were moderately differentiated, as reflected by the Hudson's F(ST) value of 0.14, and they represent regions on LG7 with strikingly elevated F(ST) values. Seven-fold reduction in heterozygosity and two times increase in LD (r²) were observed in the same region for the round-fruited sub-gene pool. Outlier test suggested the locus LX3405 on LG7 to be a candidate site under selection. Comparative genomic analysis revealed that the cucumber genome region syntenic to the high FST island on LG7 harbors an ortholog of the tomato fruit shape gene OVATE. Our results point to a bright future of applying RAD-Seq to population genomic studies for non-model species even under low-to-medium sequencing efforts. The genomic resources provide valuable information for cucurbit genome research.

QTL mapping and epistatic interaction analysis in asparagus bean for several characterized and novel horticulturally important traits.

BACKGROUND: Asparagus bean (Vigna. unguiculata. ssp sesquipedalis) is a subspecies and special vegetable type of cowpea (Vigna. unguiculata L. Walp.) important in Asia. Genetic basis of horticulturally important traits of asparagus bean is still poorly understood, hindering the utilization of targeted, DNA marker-assisted breeding in this crop. Here we report the identification of quantitative trait loci (QTLs) and epistatic interactions for four horticultural traits, namely, days to first flowering (FLD), nodes to first flower (NFF), leaf senescence (LS) and pod number per plant (PN) using a recombinant inbred line (RIL) population of asparagus bean. RESULTS: A similar genetic mode of one major QTL plus a few minor QTLs was found to dominate each of the four traits, with the number of QTLs for individual traits ranging from three to four. These QTLs were distributed on 7 of the 11 chromosomes. Major QTLs for FLD, NFF and LS were co-localized on LG 11, indicative of tight linkage. Genome wide epistasis analysis detected two and one interactive locus pairs that significantly affect FLD and LS, respectively, and the epistatic QTLs for FLD appeared to work in different ways. Synteny based comparison of QTL locations revealed conservation of chromosome regions controlling these traits in related legume crops. CONCLUSION: Major, minor, and epistatic QTLs were found to contribute to the inheritance of the FLD, NFF, LS, and PN. Positions of many of these QTLs are conserved among closely related legume species, indicating common mechanisms they share. To our best knowledge, this is the first QTL mapping report using an asparagus bean × asparagus bean intervarietal population and provides marker-trait associations for marker-assisted approaches to selection.

Deep sequencing-based analysis of the Cymbidium ensifolium floral transcriptome.

Cymbidium ensifolium is a Chinese Cymbidium with an elegant shape, beautiful appearance, and a fragrant aroma. C. ensifolium has a long history of cultivation in China and it has excellent commercial value as a potted plant and cut flower. The development of C. ensifolium genomic resources has been delayed because of its large genome size. Taking advantage of technical and cost improvement of RNA-Seq, we extracted total mRNA from flower buds and mature flowers and obtained a total of 9.52 Gb of filtered nucleotides comprising 98,819,349 filtered reads. The filtered reads were assembled into 101,423 isotigs, representing 51,696 genes. Of the 101,423 isotigs, 41,873 were putative homologs of annotated sequences in the public databases, of which 158 were associated with floral development and 119 were associated with flowering. The isotigs were categorized according to their putative functions. In total, 10,212 of the isotigs were assigned into 25 eukaryotic orthologous groups (KOGs), 41,690 into 58 gene ontology (GO) terms, and 9,830 into 126 Arabidopsis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 9,539 isotigs into 123 rice pathways. Comparison of the isotigs with those of the two related orchid species P. equestris and C. sinense showed that 17,906 isotigs are unique to C. ensifolium. In addition, a total of 7,936 SSRs and 16,676 putative SNPs were identified. To our knowledge, this transcriptome database is the first major genomic resource for C. ensifolium and the most comprehensive transcriptomic resource for genus Cymbidium. These sequences provide valuable information for understanding the molecular mechanisms of floral development and flowering. Sequences predicted to be unique to C. ensifolium would provide more insights into C. ensifolium gene diversity. The numerous SNPs and SSRs identified in the present study will contribute to marker development for C. ensifolium.

Functional analysis of FLOWERING LOCUS T orthologs from spring orchid (Cymbidium goeringii Rchb. f.) that regulates the vegetative to reproductive transition.

The FLOWERING LOCUS T (FT) gene plays crucial roles in regulating the vegetative-to-reproductive phase transition. The FT-like gene of spring orchid (Cymbidium goeringii Rchb. f.), CgFT, was isolated and characterized. CgFT mRNA was detected in leaves, pseudobulb, and flowers. In flowers, CgFT was expressed more in young flower buds than in mature flowers, and was predominantly expressed in young ovary and sheath. Seasonal expression analysis in leaves of a three-year-old spring orchid showed that a large increase in transcription, which started on June 20 for CgFT. We propose that the increased transcription in the middle of June marks the beginning of flower induction in this species. The ectopic expression of CgFT in transgenic tobacco plants showed novel phenotypes by flowering earlier than wild-type plants. Further analysis of the flowering time-related genes indicated that the expression of LEAFY, APETALLA1, FRUITFULL and SEPALLATA1 were significantly upregulated in 35S::CgFT transgenic tobacco plants. These results indicated that CgFT is a putative FT homolog in spring orchid that regulates flower transition, similar to its homolog in Arabidopsis. This study provides the first information on the spring orchid floral gene to elucidate the regulation of the flowering transition in spring orchid.

A SNP and SSR based genetic map of asparagus bean (Vigna. unguiculata ssp. sesquipedialis) and comparison with the broader species.

Asparagus bean (Vigna. unguiculata ssp. sesquipedialis) is a distinctive subspecies of cowpea [Vigna. unguiculata (L.) Walp.] that apparently originated in East Asia and is characterized by extremely long and thin pods and an aggressive climbing growth habit. The crop is widely cultivated throughout Asia for the production of immature pods known as 'long beans' or 'asparagus beans'. While the genome of cowpea ssp. unguiculata has been characterized recently by high-density genetic mapping and partial sequencing, little is known about the genome of asparagus bean. We report here the first genetic map of asparagus bean based on SNP and SSR markers. The current map consists of 375 loci mapped onto 11 linkage groups (LGs), with 191 loci detected by SNP markers and 184 loci by SSR markers. The overall map length is 745 cM, with an average marker distance of 1.98 cM. There are four high marker-density blocks distributed on three LGs and three regions of segregation distortion (SDRs) identified on two other LGs, two of which co-locate in chromosomal regions syntenic to SDRs in soybean. Synteny between asparagus bean and the model legume Lotus. japonica was also established. This work provides the basis for mapping and functional analysis of genes/QTLs of particular interest in asparagus bean, as well as for comparative genomics study of cowpea at the subspecies level.