K. V. Ravishankar

Assessment of genetic relatedness among mango cultivars of India using RAPD markers

Summary DNA-based RAPD (Random Amplification of Polymorphic DNA) markers have been used extensively to study genetic relationships in a number of fruit crops. A wide genetic diversity exists in the mango fruit in India. Present day commercial cultivars originated mainly from this subcontinent. In this study, 18 commercial mango cultivars, traditionally grown in western, southern, northern and eastern parts of India, were selected to assess genetic relatedness. Total genomic DNA was extracted and subjected to RAPD analysis using 30 arbitrary 10-mer primers. Of these, 27 primers amplified mango genomic DNA. None of these primers produced unique band pattern for each cultivar. RAPD data were used to calculate a squared Euclidean distance matrix, and based on this cluster analysis was done using a minimum variance algorithm. Cluster analysis clearly showed two groups—the first consisting of western, northern and eastern mango cultivars and the second group consisting of southern cultivars. From the analysis of results, it appears the majority of mango cultivars originated from a local mango genepool and were domesticated later.

Development of new microsatellite markers from Mango (Mangifera indica) and cross-species amplification

PREMISE OF THE STUDY Microsatellite markers were developed and characterized to assess the genetic diversity among mango (Mangifera indica) cultivars and to test their amplification in closely related species. METHODS AND RESULTS Thirty-six microsatellite (simple sequence repeats; SSR) loci were isolated by a microsatellite-enriched partial genomic library method. Primers designed for these loci were characterized using 30 diverse mango cultivars. The number of alleles ranged from 3 to 19 with an average of 9.2 alleles per locus. Polymorphic information content values ranged from 0.185 to 0.920 with a mean of 0.687. The total value for the probability of identity was 2.42 × 10(-31). CONCLUSIONS The newly identified SSRs would be useful in genetic diversity studies, finger-printing, and mapping. Loci from five related species, M. odorata, M. anadamanica, M. zeylanica, M. camptosperma, and M. griffithii, were successfully amplified using these SSR primers, showing their potential utility across species.

Genetic diversity and population structure analysis of mango (Mangiferaindica) cultivars assessed by microsatellite markers

Key messageSSR marker characterization of Indian mango cultivars revealed two distinct groups of populations with geographical affiliation. Six SSR loci with low PI have been identified as universal markers for mango characterization.AbstractIn this study, genetic diversity and population structure of mango cultivars were assessed by employing fourteen simple sequence repeat markers, with high polymorphic information content. A set of 387 mango accessions from different regions of India was used. Model-based structure analysis revealed the presence of two subpopulations comprising the cultivars from ‘South-West’ region and ‘North-East’ region. A similar clustering pattern was observed in the dendrogram analysis, with two major groups identified that were further sub-grouped based on their genetic relatedness. Analysis of molecular variance showed a significant variance component among and within mango sub-populations derived from the structure analysis. The proportion of genetic differentiation among individuals within the two populations was found to be significant with a FST value of 0.248. The extent of genetic diversity was found to be higher in case of ‘South and West’ population than ‘North and East’ population. Six SSR loci with low probability of identity values have been chosen as universal markers for mango characterization. Our results highlight genetic diversity encompassed by mango cultivars and genetic distinctness of ‘South-West’ and ‘North-East’ region cultivars.

Differential expression of carotenoid biosynthetic pathway genes in two contrasting tomato genotypes for lycopene content

Tomato (Solanum lycopersicum L.) is one of the model plant to study carotenoid biosynthesis. In the present study, the fruit carotenoid content were quantified at different developmental stages for two contrasting genotypes, viz. IIHR-249-1 and IIHR-2866 by UPLC. Lycopene content was high in IIHR-249-1 (19.45 mg/100 g fresh weight) compared to IIHR-2866 (1.88 mg/100 g fresh weight) at the ripe stage. qPCR was performed for genes that are involved in the carotenoid biosynthetic pathway to study the difference in lycopene content in fruits of both the genotypes. The expression of Phytoene synthase (PSY) increased by 36-fold and Phytoene desaturase (PDS) increased by 14-fold from immature green stage to ripe stage in IIHR-249-1. The expression of Chloroplast lycopene β-cyclase (LCY-B) and Chromoplast lycopene β cyclase (CYC-B) decreased gradually from the initial stage to the ripe stage in IIHR-249-1. IIHR 249-1 showed 3- and 1.8-fold decrease in gene expression for Chloroplast lycopene β-cyclase (LCY-B) and Chromoplast lycopene β-cyclase (CYC-B) .The F2 hybrids derived from IIHR-249-1 and IIHR-2866 were analysed at the ripe stage for lycopene content. The gene expression of Chloroplast lycopene β-cyclase (LCY-B) and Chromoplast lycopene β-cyclase (CYC-B) in high and low lycopene lines from F2 progenies also showed the decrease in transcript levels of both the genes in high lycopene F2 lines. We wish to suggest that the differential expression of lycopene β-cyclases can be used in marker-assisted breeding.

Development of SCAR marker for sex identification in dioecious Garcinia gummi-gutta

Key messageWe have developed sex-specific SCAR marker for the identification of dioeciousGarcinia gummi-gutta(L.), which is useful for the selection ofG. gummi-guttaat seedling stage and for plantation programmes.AbstractGarcinia gummi-gutta (L.) Robs. is a dioecious fruit yielding tree, which is naturally distributed as well as cultivated in the orchards in Western Ghat regions of India. A sex-linked DNA fragment was identified in Garcinia gummi-gutta (L.) Robs. by screening 150 randomly amplified polymorphic DNA primers and only one of them (OPBD20) showed different amplification band pattern associated with sex type. This sex-linked fragment was converted into male-specific sequence-characterized amplified region (SCAR) marker, CAM-566. The primers deigned in this study (OPBD20F and OPBD20R) correctly differentiated 12 male and 12 female plants at high annealing temperatures. Thus, a 556-bp band was amplified in male samples but not in female ones. Nevertheless, it should be noted that the fragments from both sexes were amplified at relatively low annealing temperatures. Additionally, the developed SCAR marker successfully identified the sexes of ten sex-unknown samples. Therefore, it can be used as an effective, convenient and reliable tool for sex determination in such dioecious species.

Development of male-specific SCAR marker in Garcinia morella (Gaertn.) Desr.

Garcinia morella (Gaertn.) Desr. (family Clusiaceae) is popularly known as ‘Indian gamboge’ is a fruit yielding tree of tropical rain forests of Western Ghats of India (Anonymous 1956). It also occurs in Sri Lanka and Indo-China Himalayan regions. It is a multipurpose tree grown as a plantation crop along with Garcinia indica (Kokum) and Garcinia cambogia (Malabar tamarind). The fruit rinds are used as a condiment and for garnish. Bioactive compounds such as moreollin (Subba Rao et al. 1978), gambogic acid (Tang et al. 2011) have been isolated from the fruits and the bark, respectively, and evaluated for their antibiotic and anticancer properties. The trees are dioecious and the distinction between male and female trees can be made only at the flowering stage, after 10–12 years. It is cultivated as a plantation crop and the sex determination in this plant at an early juvenile stage will be useful for planning the male and female tree ratio in the orchards and it also enables the tree improvement programme. Recently, molecular tools were employed in dioecious taxa for early identification of sex and understanding the developmental and the evolutionary pathways of sexual dimorphism. Specific molecular markers can be deduced from unique, single-copy segments of the genome and can be considered codominant and can be used in sex determination. Sequence characterized markers (SCAR) which are based on randomly amplified polymorphic DNA (RAPD) analysis are locus-specific, more reliable and more reproducible for molecular identification (Paran and Michelmore 1993). SCAR marker linked to sex-specific genes have beensuccessfully used in sex identification of many dioecious plants including Carica papaya (Bedoya and Nunez 2007), Phoenix dactylifera (Dhawan et al. 2013). In this

Development of SSR markers based on a survey of genomic sequences and their molecular analysis in banana (Musa spp.)

Summary Musa (Family Musaceae) is economically the fourth most-important crop after rice, wheat, and maize. Present-day edible banana evolved by intra- and inter-specific hybridisation of two wild species M. acuminata and M. balbisiana. A total of 229 primers were designed based on a survey of the genomic sequence data available on Musa spp. in the National Center for Biotechnology Information database. Twenty-six microsatellite (SSR) markers were amplified in 15 banana accessions. These 26 SSR markers revealed a total of 88 alleles, ranging from two-to-six alleles per locus, with an average of 3.38 alleles per locus. Genetic analysis resulted in observed heterozygosity (Ho) values ranging from 0.06 – 0.80, with a mean of 0.40. Polymorphic information content (PIC) values ranged from 0.21 – 0.77, with a mean of 0.47. A dendogram based on the SSR data grouped the 15 different banana accessions into three groups. These new SSR markers may be of value for future characterisation of banana germplasm, gene mapping, and genetic analysis.

Current Status of Banana Genome in the Age of Next Generation Sequencing

Banana (Musa spp.) is the “queen of tropical fruits.” It is one of the major staple fruits in many countries. The banana improvement program is challenging due to its complex evolutionary events, human selection, and parthenocarpy. Musa acuminata and Musa balbisiana are the progenitor species for majority of the modern cultivated bananas. The only way to accelerate the banana breeding program is to understand its genome and employing marker-assisted selection. Recently sequencing of the 523 Mb genome of a Musa acuminata – DH-Pahang provided a great fillip to the banana improvement program. Banana genome sequencing revealed the presence of around 36,000 protein coding regions, and transposable elements accounted for more than half of the genome. Earlier attempts of Bacterial artificial chromosome (BAC) sequencing of both these species showed a high degree of collinearity. In this chapter, we have summarized the current status of our understanding of the banana genome with respect to classical linkage mapping approach as well as modern next-generation sequencing approach.

Isolation and characterization of microsatellite markers in Garcinia gummi-gutta by next-generation sequencing and cross-species amplification

Garcinia gummi-gutta (L.) Roxb. (Clusiaceae) is an endemic, semidomesticated, fruit-yielding tree species distributed in the Western Ghats of India and Sri Lanka. Various bioactive phytochemicals, such as garcinol, benzophenones and xanthones are isolated from G. gummi-gutta and have shown antibacterial, antiviral and antioxidant activities. We sequenced the total genomic DNA using Illumina Hiseq 2000 platform and examined 241,141,804 bp high quality data, assembled into 773,889 contigs. In these contigs, 27,313 simple-sequence repeats (SSRs) were identified, among which mononucleotide repeats were predominant (44.98%) followed by dinucleotide and trinucleotide repeats. Primers were designed for 9964 microsatellites among which 32 randomly selected SSR primer pairs were standardized for amplification. Polymerase chain reaction (PCR) amplification of genomic DNA in 30 G. gummi-gutta genotypes revealed polymorphic information content (PIC) across all 32 loci ranging from 0.867 to 0.951, with a mean value of 0.917. The observed and expected heterozygosity ranged from 0.00 to 0.63 and 0.896 to 0.974, respectively. Alleles per locus ranged from 12 to 27. This is the first report on the development of genomic SSR markers in G. gummi-gutta using next-generation sequencing technology. The genomic SSR markers developed in this study will be useful in identification, mapping, diversity and breeding studies.

Barrier against water loss: relationship between epicuticular wax composition, gene expression and leaf water retention capacity in banana

In the present study we examined 13 banana (Musa spp.) genotypes belonging to different genomic groups with respect to total leaf cuticular wax concentration, chemical composition, carbon chain length and their relationship with leaf water retention capacity (LWRC). A positive correlation between epicuticular wax content and LWRC clearly indicated that the cuticular wax plays an important role in maintaining banana leaf water content. The classification of hexane soluble cuticular wax components into different classes based on functional group and their association with LWRC showed that alcohol and ester compounds have a positive correlation. Further, the compounds with >C28 carbon chain length had a positive correlation with LWRC, indicating the role of longer carbon chain length in maintaining the water status of banana leaves. Also, the gene expression analysis showed higher expression of the wax biosynthetic genes FATB and KCS11 in higher wax load genotypes whereas lower expression was seen in low wax banana genotypes. Here, we report for the first time the compositional variations of cuticular wax in different banana genotypes, followed by their association with leaf water retention capacity. The results were also supported by variation in gene expression analysis of cuticular wax biosynthetic genes - FATB and KCS11.