Cross species/genera transferability of simple sequence repeat markers, genetic diversity and population structure analysis in gladiolus (Gladiolus × grandiflorus L.) genotypes.

Background: Genetic analysis of gladiolus germplasm using simple sequence repeat (SSR) markers is largely missing due to scarce genomic information. Hence, microsatellites identified for related genera or species may be utilized to understand the genetic diversity and assess genetic relationships among cultivated gladiolus varieties. Methods: In the present investigation, we screened 26 genomic SSRs (Gladiolus palustris, Crocus sativus, Herbertia zebrina, Sysirinchium micranthum), 14 chloroplast SSRs (Gladiolus spp., chloroplast DNA regions) and 25 Iris Expressed Sequence Tags (ESTs) derived SSRs across the 84 gladiolus (Gladiolus × grandiflorus L.) genotypes. Polymorphic markers detected from amplified SSRs were used to calculate genetic diversity estimates, analyze population structure, cluster analysis and principal coordinate analysis (PCoA). Results: A total of 41 SSRs showed reproducible amplification pattern among the selected gladiolus cultivars. Among these, 17 highly polymorphic SSRs revealed a total of 58 polymorphic alleles ranging from two to six with an average of 3.41 alleles per marker. Polymorphic information content (PIC) values ranged from 0.11 to 0.71 with an average value of 0.48. A total of 4 SSRs were selectively neutral based on the Ewens-Watterson test. Hence, 66.66% of Gladiolus palustris, 48% of Iris spp. EST, 71.42% of Crocus sativus SSRs showed cross-transferability among the gladiolus genotypes. Analysis of genetic structure of 84 gladiolus genotypes revealed two subpopulations; 35 genotypes were assigned to subpopulation 1, 37 to subpopulation 2 and the remaining 12 genotypes could not be attributed to either subpopulation. Analysis of molecular variance indicated maximum variance (53.59%) among individuals within subpopulations, whereas 36.55% of variation among individuals within the total population. The least variation (9.86%) was noticed between two subpopulations. Moderate (FST = 0.10) genetic differentiation between two subpopulations was observed. The grouping pattern of population structure was consistent with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on simple matching dissimilarity coefficient and PCoA. Conclusion: SSR markers from the present study can be utilized for cultivar identification, conservation and sustainable utilization of gladiolus genotypes for crop improvement. Genetic relationships assessed among the genotypes of respective clusters may assist the breeders in selecting desirable parents for crossing.

Production of Haploids and Doubled Haploids in Marigold (Tagetes spp.) Using Anther Culture.

Homozygous parental lines are indispensable for commercial hybrid seed production in many ornamental and vegetable crops. The in vitro induction of haploids and doubled haploids (DHs) through gametic embryogenesis is an effective approach for single-step development of complete homozygous lines from heterozygous donor plants. Anther culture is one of the most popular and widely employed techniques for development of haploids. Here we describe the detailed protocol for rapid and successful induction of haploids in Tagetes spp. using in vitro androgenesis approach. In this protocol, we have provided the comprehensive details of various steps of anther culture in marigold right from the growing of donor plants, selection of buds, pretreatment, embryogenesis and regeneration to ploidy analysis, and chromosome doubling for development of DHs.

Improvement in molecular detection of phytoplasma associated with rose by selection of suitable primers and development of a multiplex PCR assay.

Sensitive and effective phytoplasma DNA amplification in symptomatic rose cultivars is a long unresolved problem. In the present study, improvement in standardization for PCR assay for phytoplasma detection was established with rose samples by selection of various combinations of nested primer pairs of 16S ribosomal gene and secA gene. CTAB DNA extraction method was slightly modified by adding 2% polyvinyl pyrrolidone and increased the isopropanol volume which yielded better quality DNA. Best amplification results were achieved in nested PCR assay employing P1/P7, R16mF2/R16mR2 and R16F2n/R16R2, P1/P7 and R16mF2/R16mR2, and R16mF2/R16mR2 and fU5/rU3 primer pairs. Besides, a multiplex PCR assay was also developed and optimized for consistent identification of phytoplasma in rose samples by employing primer pairs of 16S rRNA and secA genes together in a single PCR reaction by optimizing annealing temperature at 55 °C.

Characterization of Indian bred rose cultivars using morphological and molecular markers for conservation and sustainable management.

Rose (Rosa × hybrid L.) is one of the most important commercial ornamental crops cultivated worldwide for its beauty, fragrance and nutraceutical values. Characterization of rose germplasm provides precise information about the extent of diversity present among the cultivars. It also helps in cultivar identification, intellectual property right protection, variety improvement and genetic diversity conservation. In the present study, 109 Indian bred rose cultivars were characterized using 59 morphological and 48 SSR markers. Out of 48 SSRs used, 31 markers exhibited polymorphism and 96 alleles were identified with an average of 3.9 alleles per locus. Nei's expected heterozygosity value of each locus ranged from 0.08 (with SSR ABRII/RPU32) to 0.78 (SSR Rh58). The similarity coefficient values ranged from 0.42 to 0.90 which indicated presence of moderated diversity among Indian cultivars. The neighbor-joining tree based on morphological data grouped the cultivars into two major clusters and several minor clusters based on their morphological resemblance. However, UPGMA dendrogram constructed using matching coefficient values grouped the cultivars into eight different clusters. Interpopulation analysis revealed higher genetic similarities between Hybrid Tea and Floribunda cultivars. An analysis for presence of population sub-structure grouped the Indian cultivars into eight different genetic groups. Analysis of molecular variance revealed apportioning of 97.59% of the variation to within subgroup diversity and 3.07% to between the cultivar groups. We have demonstrated here successful utilization of robust SSR to distinguish cultivars and assess genetic diversity among Indian bred rose cultivars. The information provided here is useful for cultivar identification and protection, cultivar improvement and genetic diversity conservation.