BSA and molecular markers screening for salt stress tolerant mutant of Petunia obtained in in vitro culture

ABSTRACT: In this study, we performed BSA to identify genetic markers linked to salt tolerance. We tested the genetic diversity among four bulked DNA samples of EMS induced mutant clones and one bulked DNA sample of non-mutated clone of Petunia for salt tolerance in in vitro callus cultures using RAPD and ISSR markers. Out of the 36 RAPD and 16 ISSR primers identified, 25 and 13 were effectively used to amplify genomic DNA of all the five bulked samples, respectively. In total, 114 RAPD amplifications products were obtained, of which 28% were polymorphic and 2% were genotype-specific bands. Out of the 64 ISSR amplification products obtained, 51% were polymorphic and 1% was genotype-specific bands. Results of this study indicated the existence of two patterns of distorted segregation among the studied markers. The first one indicates the differences between non-mutated clones of Petunia and its putative mutants. The second one was observed only between putative mutants and putative mutants tested for salt tolerance in in vitro culture. Both RAPD and ISSR analysis successfully detected the association with changes induced by chemical mutagenesis and salinity. Furthermore, our results indicate that BSA method can be useful in the rapid detection of molecular markers for further marker-assisted selection.

Genetic diversity and differentiation of Pinus sylvestris L. from the IUFRO 1982 provenance trial revealed by AFLP analysis

DNA markers have become effective tools in genetic diversity studies of forest trees. However, molecular marker analyses are associated with laborious and costly effort. One of the possibilities to overcome these constraints is to analyze bulked samples per population, rather than individual plants. We have used bulked DNA-based AFLP analysis to investigate genetic variations in Pinus sylvestris L. (Scots pine) from the IUFRO 1982 provenance trial in K?rnik (western Poland). Four AFLP primer combinations yielded a total of 309 bands, of which 208 (67.31%) were polymorphic. Thirty-six (11.65%) unique alleles were deployed randomly among the populations. Estimated genetic diversity and differentiation was high, as expressed by He = 0.238 and I = 0.356, and by genetic distance values which ranged from 0.154 to 0.363. A geographic pattern of interpopulation differentiation was observed, pointing to the individual character of populations from northeastern Europe. In the light of available data, we discuss the influence of historical migration routes, gene flow and human activity on observed genetic diversity and differentiation of Scots pine in Europe. Our results indicate that the AFLP method applied to DNA templates extracted from bulked leaf samples provides an efficient approach to elucidate genetic diversity and relationships among Scots pine populations.

Genetic variation and relationship of alfalfa populations and their progenies based on RAPD markers

The aim of investigation was to evaluate genetic variation and relationship among alfalfa populations and their offspring, with minimal cost, by using DNA marker analysis. RAPD analysis was performed on bulked DNA samples of five alfalfa parental populations and their progenies: 20 F1 populations from reciprocal diallel crosses and five S1 populations from self-pollination. Twenty primers generated 217 bands, ranging in size from 300 to 6000 bp, with the average number of bands per primer of 10.85 and polymorphism information content of 0.246. Percentage of polymorphic loci, effective number of alleles, expected heterozygosity and Shannon’s information index were used to estimate genetic variation. Higher diversity was observed in F1 progeny populations, while genetic variation in parental populations and S1 progenies remained similar. The genetic relatedness of alfalfa populations was analysed by UPGMA and Bayesian model-based clustering approach. In both types of analysis selfpollinated progenies were grouped. Furthermore, the hybrid offspring where Zuzana, and RSI 20 were maternal parents were placed in separate groups. The results indicate that use of RAPD markers on bulked DNA samples can be fast and cost-effective way for differentiation of alfalfa parental populations and their offspring, as well as for evaluation of their genetic relationships.

Application of RAPD markers in hybrid verification in coconut

Coconut palms are classified into two major types, viz., 'talls' and 'dwarfs', which mainly differ in their pollination behavior of cross- and self-pollination, respectively. Due to this difference, getting true-to-type progenies of desirable tall and dwarf cultivars has always been a challenge. The conventional practice of selection of seedlings based solely on morphological traits often results in selection of out-crossed seedlings and undesirable off-types. In the present investigation, RAPD markers for the tall/dwarf trait were identified in coconut using a bulked DNA approach. Screening of tall and dwarf palm bulk DNA with 200 primers revealed a RAPD primer OPBA3 which was able to clearly differentiate both the tall and dwarf bulks. For validation, the primer was used to screen individual tall and dwarf coconut palms representing different geographic regions. The primer was also used to screen the parents and validate hybrids of Dwarf x Tall crosses.

Regulation of pepper fruit color, chloroplasts development and their importance in fruit quality

Pepper exhibits large natural variation in chlorophyll content in the immature fruit. To dissect the genetic and molecular basis of this variation, we conducted QTL mapping for chlorophyll content in a cross between light and dark green-fruited parents, PI 152225 and 1154. Two major QTLs, pc1 and pc10, that control chlorophyll content by modulation of chloroplast compartment size in a fruit-specific manner were detected in chromosomes 1 and 10, respectively. The pepper homolog of GOLDEN2- LIKE transcription factor (CaGLK2) was found as underlying pc10, similar to its effect on tomato fruit chloroplast development. A candidate gene for pc1was found as controlling chlorophyll content in pepper by the modulation of chloroplast size and number. Fine mapping of pc1 aided by bulked DNA and RNA-seq analyses enabled the identification of a zinc finger transcription factor LOL1 (LSD-One-Like 1) as a candidate gene underlying pc1. LOL1 is a positive regulator of oxidative stress- induced cell death in Arabidopsis. However, over expression of the rice ortholog resulted in an increase of chlorophyll content. Interestingly, CaAPRR2 that is linked to the QTL and was found to affect immature pepper fruit color in a previous study, did not have a significant effect on chlorophyll content in the present study. Verification of the candidate's function was done by generating CRISPR/Cas9 knockout mutants of the orthologues tomato gene, while its knockout experiment in pepper by genome editing is under progress. Phenotypic similarity as a consequence of disrupting the transcription factor in both pepper and tomato indicated its functional conservation in controlling chlorophyll content in the Solanaceae. A limited sequence diversity study indicated that null mutations in CaLOL1 and its putative interactorCaMIP1 are present in C. chinensebut not in C. annuum. Combinations of mutations in CaLOL1, CaMIP1, CaGLK2 and CaAPRR2 are required for the creation of the extreme variation in chlorophyll content in Capsicum.

Development of AFLP markers linked to stem nematode resistance gene in sweet potato (Ipomoea batatas (L.) Lam.)

Amplified fragment length polymorphism (AFLP) markers linked to the stem nematode resistance gene were developed in sweet potato (Ipomoea batatas (L.) Lam.). Using bulked segregant analysis (BSA), 800 AFLP primer combinations were screened in the resistant and susceptible bulked DNA from the 186 progeny of an F1 single-cross population of Xu781 (resistant parent)×Xushu18 (susceptible parent), and 245 of these AFLP primers showed polymorphic bands between resistant and susceptible DNA. Primer combinations detecting polymorphism between the two bulks were used to screen the parents and eight individuals from each of the bulks. The results showed that E2M23 and E33M20 produced a specific band of about 500 bp and 200 bp in length, respectively, in the resistant plants but not in the susceptible plants, suggesting that the markers named E2M23500 and E33M20200 linked to a gene for stem nematode resistance. Amplified analysis of the 186 F1 individuals indicated that the genetic distance between these two markers and the stem nematode resistance gene was 6.9 cM and 11.1 cM, respectively, measured with Mapmaker 3.0. These two AFLP markers were used to identify ten sweet potato varieties planted widely in China and the results were consistent with those of conventional resistance identification, indicating that the two markers can be used in molecular marker-assisted breeding for stem nematode resistance in the sweet potato.

Genetic Mapping and Functional Analysis of the Tomato Bs4 Locus Governing Recognition of the Xanthomonas campestris pv. vesicatoria AvrBs4 Protein

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper (Capsicum spp.) and tomato (Lycopersicon spp.). Analysis of 17 different Lycopersicon accessions with avrBs4-expressing X. campestris pv. vesicatoria strains identified 15 resistant and two susceptible tomato genotypes. Genetic analysis revealed that AvrBs4 recognition in tomato is governed by a single locus, designated Bs4 (bacterial spot resistance locus no. 4). Amplified fragment length polymorphism and bulked DNA templates from resistant and susceptible plants were used to define a 2.6-cM interval containing the Bs4 locus. A standard tomato mapping population was employed to localize Bs4- linked markers on the short arm of chromosome 5. Investigation of X. campestris pv. vesicatoria hrp mutant strains revealed that AvrBs4 secretion and avirulence activity are hrp dependent. Agrobacterium-based delivery of the avrBs4 gene into tomato triggered a plant response that phenotypically resembled the hypersensitive response induced by avrBs4-expressing X. campestris pv. vesicatoria strains, suggesting symplastic perception of the avirulence protein. Mutations in the avrBs4 C-terminal nuclear localization signals (NLSs) showed that NLSs are dispensable for Bs4-mediated recognition. Our data suggest that tomato Bs4 and pepper Bs3 employ different recognition modes for detection of the highly homologous X. campestris pv. vesicatoria avirulence proteins AvrBs4 and AvrBs3.