Reaping the Benefits of Next-generation Sequencing Technologies for Crop Improvement — Solanaceae

Next-generation sequencing (NGS) technologies make possible the sequencing of the whole genome of a species decoding a complete gene catalogue and transcriptome to allow the study of expression pattern of entire genes. The huge data generated through whole genome and transcriptome sequencing not only provide a basis to study variation at gene sequence (such as single-nucleotide polymorphism and InDels) and expression level but also help to understand the evolutionary relationship between different crop species. Furthermore, NGS technologies have made possible the quick correlations of phenotypes with genotypes in different crop species, thereby increasing the precision of crop improvement. The Solanaceae family represents the third most economically important family after grasses and legumes due to high nutritional components. The current advances in NGS technology and their application in Solanaceae crops made several progresses in the identification of genes responsible for economically important traits, development of molecular markers, and understanding the genome organization and evolution in Solanaceae crops. The combination of high-throughput NGS technologies with conventional crop breeding has been shown to be promising in the Solanaceae translational genomics research. As a result, NGS technologies has been seen to be adopted in a large scale to study the molecular basis of fruit and tuber development, disease resistance, and increasing quantity and quality of crop production

Study of polyembryony and development of molecular markers for identification of zygotic and nucellar seedlings in Khasi mandarin (Citrus reticulata Blanco)

The objective of this work was to evaluate the occurrence of polyembryonic seedlings and other morphological parameters in Khasi mandarin during three harvest years and to identify zygotic (sexual) seedlings from nucellar (asexual) ones grown under in-vitro conditions using molecular markers. Embryos from 27 polyembryonic and 7 monoembryonic seeds of Khasi mandarin were grown in-vitro. DNA from seedlings and mother parent was analyzed using 16 ISSR and 5 RAPD primers, of which 4 ISSR and a set of 3 RAPD primers were effective to identify zygotic or nucellar origin of the seedlings. In-vitro culture enables maximum embryos of each seed to grow, favouring the origin of seedlings to be identified as zygotic. Among 69 tested individuals, 37 zygotic and 32 nucellar seedlings were recognized. In polyembryonic and monoembryonic seeds, 59.6% and 42.8% of the seedlings, respectively, have the sexual origin. Morphological characteristics of seeds and the seedlings generated varied significantly and were not correlated with polyembryony except for the clutch size and the number of branches. Polyembryonic seeds in the cultivar are high, ranging from 50.0%, 55.5% to 83.3% over three harvest years with more clutch size and the possibility of obtaining zygotic plants from them is high. In polyembryonic seeds not all zygotic seedlings were produced by small embryos located at the micropyle. Identification of zygotic seedlings by ISSR and RAPD markers in Khasi mandarin cultivar is efficient and reliable at an early developmental stage

Analysis of bioactive components in Ghost chili (<i>Capsicum chinense</i>) for antioxidant, genotoxic, and apoptotic effects in mice

<p>Apart from using traditionally in culinary preparations, chili peppers are also important constituents of herbal medicines. Although the bioactive components are studied mostly in the fruits of <i>Capsicum annuum</i>, no such study reports till date is available for Ghost chili (<i>C. chinense</i>) from North East India. Therefore, the aim of this study was to carry out an analysis of the bioactive constituents in the naturally occurring hottest chili Ghost chili (<i>C. chinense</i>), and evaluate its antioxidant, pro/anti-genotoxic, and apoptotic effects in <i>in vitro</i> and <i>in vivo</i> models. Three different antioxidant assays showed that lower doses of Ghost chili extract showed higher DNA protective and antioxidant activities. Furthermore, the administration for 7 alternate days into 6 week old Swiss albino mice showed that the lower doses (50 and 100 mg/kg bw) reduced DMBA induced genotoxicity beside significantly enhancing the activities of hepatic antioxidant enzymes, while higher dose (200 mg/kg bw) induced genotoxic effect in bone marrow cells. The administration of higher dose (200 mg/kg bw) also induced apoptosis and upregulation of <i>Bax</i> (pro) and downregulation of <i>Bcl-2</i> (anti) apoptotic genes. Dose dependent increase of apoptosis was also observed in Hep G2 and Hep 3B liver cancer cell lines. Our findings in the present study suggest that low doses of <i>C. chinense</i> can exert cancer chemopreventive effects. The induction of apoptosis in both cancer cell lines and mouse bone marrow cells, and up-regulation of proapoptotic genes suggests that the higher dose of <i>C. chinense</i> can be used for targeted cancer therapy.</p

MiR1885 Regulates Disease Tolerance Genes in Brassica rapa during Early Infection with Plasmodiophora brassicae

Clubroot caused by Plasmodiophora brassicae is a severe disease of cruciferous crops that decreases crop quality and productivity. Several clubroot resistance-related quantitative trait loci and candidate genes have been identified. However, the underlying regulatory mechanism, the interrelationships among genes, and how genes are regulated remain unexplored. MicroRNAs (miRNAs) are attracting attention as regulators of gene expression, including during biotic stress responses. The main objective of this study was to understand how miRNAs regulate clubroot resistance-related genes in P. brassicae-infected Brassica rapa. Two Brassica miRNAs, Bra-miR1885a and Bra-miR1885b, were revealed to target TIR-NBS genes. In non-infected plants, both miRNAs were expressed at low levels to maintain the balance between plant development and basal immunity. However, their expression levels increased in P. brassicae-infected plants. Both miRNAs down-regulated the expression of the TIR-NBS genes Bra019412 and Bra019410, which are located at a clubroot resistance-related quantitative trait locus. The Bra-miR1885-mediated down-regulation of both genes was detected for up to 15 days post-inoculation in the clubroot-resistant line CR Shinki and in the clubroot-susceptible line 94SK. A qRT-PCR analysis revealed Bra019412 expression was negatively regulated by miR1885. Both Bra019412 and Bra019410 were more highly expressed in CR Shinki than in 94SK; the same expression pattern was detected in multiple clubroot-resistant and clubroot-susceptible inbred lines. A 5′ rapid amplification of cDNA ends analysis confirmed the cleavage of Bra019412 by Bra-miR1885b. Thus, miR1885s potentially regulate TIR-NBS gene expression during P. brassicae infections of B. rapa

Mapping QTLs of resistance to head splitting in cabbage (Brassica oleracea L.var. capitata L.)

Cabbage head splitting can greatly affect both the quality and commercial value of cabbage (Brassica oleracea). To detect the genetic basis of head-splitting resistance, a genetic map was constructed using an F2 population derived by crossing “748” (head-splitting-resistant inbred line) and “747” (head-splitting-susceptible inbred line). The map spans 830.9\ua0cM and comprises 270 markers distributed in nine linkage groups, which correspond to the nine chromosomes of B. oleracea. The average distance between adjacent markers was 3.6\ua0cM. A total of six quantitative trait loci (QTLs) conferring resistance to head splitting were detected in chromosome 2, 4, and 6. Two QTLs, SPL-2-1 and SPL-4-1, on chromosomes 2 and 4, respectively, were detected in the experiments over 2\ua0years, suggesting that these two potential loci were important for governing the head-splitting resistance trait. Markers BRPGM0676 and BRMS137, which were tightly linked with head-splitting resistance, were detected in the conserved QTL SPL-2-1 region using bulked segregant analysis. Synteny analysis showed that SPL-2-1 was anchored to a 3.18-Mb genomic region of the B. oleracea genome, homologous to crucifer ancestral karyotype E block in chromosome 1 of Arabidopsis thaliana. Moreover, using a field emission scanning electron microscope, significant differences were observed between the two parental lines in terms of cell structures. Line “747” had thinner cell wall, lower cell density, larger cell size, and anomalous cell wall structure compared with the resistant line “748.” The different cell structures can provide a cytological base for assessing cabbage head splitting

Comparative Analysis of Fruit Metabolites and Pungency Candidate Genes Expression between Bhut Jolokia and Other <i>Capsicum</i> Species

<div><p>Bhut jolokia, commonly known as Ghost chili, a native <i>Capsicum</i> species found in North East India was recorded as the naturally occurring hottest chili in the world by the Guinness Book of World Records in 2006. Although few studies have reported variation in pungency content of this particular species, no study till date has reported detailed expression analysis of candidate genes involved in capsaicinoids (pungency) biosynthesis pathway and other fruit metabolites. Therefore, the present study was designed to evaluate the diversity of fruit morphology, fruiting habit, capsaicinoids and other metabolite contents in 136 different genotypes mainly collected from North East India. Significant intra and inter-specific variations for fruit morphological traits, fruiting habits and 65 fruit metabolites were observed in the collected <i>Capsicum</i> germplasm belonging to three <i>Capsicum</i> species i.e., <i>Capsicum chinense</i> (Bhut jolokia, 63 accessions), <i>C</i>. <i>frutescens</i> (17 accessions) and <i>C</i>. <i>annuum</i> (56 accessions). The pungency level, measured in Scoville Heat Unit (SHU) and antioxidant activity measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay showed maximum levels in <i>C</i>. <i>chinense</i> accessions followed by <i>C</i>. <i>frutescens</i> accessions, while <i>C</i>. <i>annuum</i> accessions showed the lowest value for both the traits. The number of different fruit metabolites detected did not vary significantly among the different species but the metabolite such as benzoic acid hydroxyl esters identified in large percentage in majority of <i>C</i>. <i>annuum</i> genotypes was totally absent in the <i>C</i>. <i>chinense</i> genotypes and sparingly present in few genotypes of <i>C</i>. <i>frutescens</i>. Significant correlations were observed between fruit metabolites capsaicin, dihydrocapsaicin, hexadecanoic acid, cyclopentane, α-tocopherol and antioxidant activity. Furthermore, comparative expression analysis (through qRT-PCR) of candidate genes involved in capsaicinoid biosynthesis pathway revealed many fold higher expression of majority of the genes in <i>C</i>. <i>chinense</i> compared to <i>C</i>. <i>frutescens</i> and <i>C</i>. <i>annuum</i> suggesting that the possible reason for extremely high pungency might be due to the higher level of candidate gene(s) expression although nucleotide variation in pungency related genes may also be involved in imparting variations in level of pungency.</p></div

Anatomic Characteristics Associated with Head Splitting in Cabbage (<i>Brassica oleracea</i> var. <i>capitata</i> L.)

<div><p>Cabbage belonging to Brassicaceae family is one of the most important vegetables cultivated worldwide. The economically important part of cabbage crop is head, formed by leaves which may be of splitting and non-splitting types. Cabbage varieties showing head splitting causes huge loss to the farmers and therefore finding the molecular and structural basis of splitting types would be helpful to breeders. To determine which anatomical characteristics were related to head-splitting in cabbage, we analyzed two contrasting cabbage lines and their offspring using a field emission scanning electron microscope. The inbred line “747” is an early head-splitting type, while the inbred line “748” is a head-splitting-resistant type. The petiole cells of “747” seems to be larger than those of “748” at maturity; however, there was no significant difference in petiole cell size at both pre-heading and maturity stages. The lower epidermis cells of “747” were larger than those of “748” at the pre-heading and maturity stages. “747” had thinner epidermis cell wall than “748” at maturity stage, however, there was no difference of the epidermis cell wall thickness in the two lines at the pre-heading stage. The head-splitting plants in the F₁ and F₂ population inherited the larger cell size and thinner cell walls of epidermis cells in the petiole. In the petiole cell walls of “747” and the F₁ and F₂ plants that formed splitting heads, the cellulose microfibrils were loose and had separated from each other. These findings verified that anomalous cellulose microfibrils, larger cell size and thinner-walled epidermis cells are important genetic factors that make cabbage heads prone to splitting.</p></div