Sequence-Tagged Site of Defense-Related Genes for Resistant/ Susceptible Eucalypt Selection to Cryptosporiopsis eucalypti

ABSTRACT Leaf spot and shoot blight caused by the fungal pathogen, Cryptosporiopsis eucalypti, is the most severe disease for eucalypt plantation in Thailand. The objective of this study was to create a set of DNA markers for resistant clone detection. Twelve clones of eucalypt were selected and screened for disease resistance using intact seedling inoculation and detached leaf techniques in the greenhouse and laboratory. Six clones, SF01, SF06, SF18, SF36, SF94 and SF98, were classified as resistance while the other six, SF03, SF07, SF14, SF16, SF70 and SF86, were susceptible clones. The sequence-tagged-site (STS) marker of pathogenesis-related (PR) genes, plant disease resistance (R) genes, and defense-related genes were analyzed in both DNAs of resistant and susceptible groups using polymerase chain reaction (PCR). Amplified DNA fragments were separated by agarose gel and polyacrylamide gel electrophoresis. The results showed that six markers (Ce1, Ce2, Ce3, Ce4, Ce5 and Ce6) from thirty five primer pairs could be used for susceptible and resistant detection of eucalypt

Chromosomal localization of the 18S-28S and 5S rRNA genes and (TTAGGG)n sequences of butterfly lizards (Leiolepis belliana belliana and Leiolepis boehmei, Agamidae, Squamata)

Chromosomal mapping of the butterfly lizards Leiolepis belliana belliana and L. boehmei was done using the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences. The karyotype of L. b. belliana was 2n = 36, whereas that of L. boehmei was 2n = 34. The 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, while the 5S rRNA genes were found in the pericentromeric region of chromosome 6 in both species. Hybridization signals for the (TTAGGG)n sequence were observed at the telomeric ends of all chromosomes, as well as interstitially at the same position as the 18S-28S rRNA genes in L. boehmei. This finding suggests that in L. boehmei telomere-to-telomere fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located or, alternatively, at the secondary constriction of chromosome 1. The absence of telomeric sequence signals in chromosome 1 of L. b. belliana suggested that its chromosomes may have only a few copies of the (TTAGGG)n sequence or that there may have been a gradual loss of the repeat sequences during chromosomal evolution

Identification of Stipules reduced, a leaf morphology gene in pea (Pisum sativum)

Pea (Pisum sativum) is one of relatively few genetically amenable plant species with compound leaves. Pea leaves have a variety of specialized organs: leaflets, tendrils, pulvini and stipules, which enable the identification of mutations that transform or affect distinct parts of the leaf. Characterization of these mutations offers insights into the development and evolution of novel leaf traits. The previously characterized morphological gene Cochleata, conferring stipule identity, was known to interact with Stipules reduced (St), which conditions stipule size in pea, but the St gene remained unknown. Here we analysed Fast Neutron irradiated pea mutants by restriction site associated DNA sequencing. We identified St as a gene encoding a C2H2 zinc finger transcription factor that is regulated by Cochleata. St regulates both cell division and cell expansion in the stipule. Our approach shows how systematic genome-wide screens can be used successfully for the analysis of traits in species for which whole genome sequences are not available

Zmiennosc loci izoenzymatycznych w kolekcji podstawowej rodzaju Pisum

Zgromadzone w krajowej kolekcji Pisum w Wiatrowie zasoby obejmują dzikie populacje, odmiany uprawne i miejscowe, wyselekcjonowane mutanty spontaniczne i indukowane oraz linie pochodzące z krzyżowań w programach hodowlanych i badawczych. Utworzono z nich kolekcję podstawową rodzaju Pisum (266 obiektów), reprezentującą dotychczas opisaną zmienność monogeniczną w genotypach typowych dla alleli, liniach testowych z genami - markerami poszczególnych chromosomów oraz z nowymi genami. Dla zwiększenia przydatności kolekcji podstawowej Pisum zgromadzonej w Wiatrowie scharakteryzowano zakres zmienności loci izoenzymatycznych. Analizowano polimorfizm 18 loci izoenzymatycznych metodą rozdziału elektroforetycznego na żelu skrobiowym. Wszystkie badane loci enzymatyczne wykazały obecność 2-4 allozymów. Niektóre z wykrytych allozymów występowały bardzo rzadko, najczęściej w populacjach należących do dzikich gatunków Pisum. Zakres obserwowanej zmienności izoenzymatycznej w poszczególnych grupach obiektów posłużył do obliczenia częstości alleli i oceny polimorfizmu każdej z grup oraz porównania zmienności pomiędzy grupami. Uzyskane wyniki wykorzystano do dyskusji na temat wykorzystania markerów izoenzymatycznych do charakterystyki genotypu obiektów kolekcyjnych.The Pisum national collection at Wiatrowo covers wild populations, culti- vars and land races, selected spontaneous and induced mutants, as well as cross derivatives from breeding and research programs. There has been constituted the core collection of Pisum genus (266 accesions), representing hitherto described monogenic variability in typical genotypes for alleles, tester lines with marker genes for particular chromosomes, and with new genes. To increase the usefulness of Pisum core collection gathered at Wiatrowo, the range of isozyme loci variability was characterized. The polymorphism of 18 isozyme loci was analyzed using electrophoretic separation on starch gel. All tested loci showed the presence of 2-4 allozymes. Some of them occurred very rarely, usually in populations belonging to the wild species of Pisum. The range of observed isozyme variability in particular groups of accesions was used for statistical calculations of allele frequency and for estimating polymorphism in each group as well as for comparison of variability among groups. The results were used for discussion on utility of isozyme markers for genotype characterization of collection accessions

Genetic evidence of multiple invasions and a small number of founders of Asian Palmyra palm (Borassus flabellifer) in Thailand

Abstract Background Borassus flabellifer or Asian Palmyra palm is an important crop for local economies in the South and Southeast Asia for its fruit and palm sugar production. Archeological and historical evidence indicated the presence of this species in Southeast Asia dating back at least 1500 years. B. flabellifer is believed to be originated in Africa, spread to South Asia and introduced into Southeast Asia through commercial routes and dissemination of cultures, however, the nature of its invasion and settlement in Thailand is unclear. Results Here, we analyzed genetic data of 230 B. flabellifer accessions across Thailand using 17 EST-SSR and 12 gSSR polymorphic markers. Clustering analysis revealed that the population consisted of two genetic clusters (STRUCTURE K = 2). Cluster I is found mainly in southern Thailand, while Cluster II is found mainly in the northeastern. Those found in the central are of an extensive mix between the two. These two clusters are in moderate differentiation (F ST = 0.066 and N M = 3.532) and have low genetic diversity (HO = 0.371 and 0.416; AR = 2.99 and 3.19, for the cluster I and II respectively). The minimum numbers of founders for each genetic group varies from 3 to 4 individuals, based on simulation using different allele frequency assumptions. These numbers coincide with that B. flabellifer is dioecious, and a number of seeds had to be simultaneously introduced for obtaining both male and female founders. Conclusions From these data and geographical and historical evidence, we hypothesize that there were at least two different invasive events of B. flabellifer in Thailand. B. flabellifer was likely brought through the Straits of Malacca to be propagated in the southern Thailand as one of the invasive events before spreading to the central Thailand. The second event likely occurred in Khmer Empire, currently Cambodia, before spreading to the northeastern Thailand

The complete chloroplast genome sequence of Asian Palmyra palm (Borassus flabellifer)

Abstract Objective Borassus flabellifer or Asian Palmyra palm is widely distributed in South and Southeast Asia and is horticultural and economic importance for its fruit and palm sugar production. However, its population is in rapid decline, and only a few genetic data are available. We sequenced the complete chloroplast (cp) genome of B. flabellifer to provide its genetic data for further utilization. Results The cp genome was obtained by Illumina sequencing and manual gap fillings providing 160,021 bp in length containing a pair of inverted repeats (IRs) with 27,256 bp. These IRs divide the genome into a large single copy region 87,444 bp and a small single copy region 18,065 bp. In total, 113 unique genes, 134 SSRs and 47 large repeats were identified. This is the first complete cp genome reported in the genus Borassus. A comparative analysis among members of the Borasseae tribe revealed that the B. flabellifer cp genome is, so far, the largest and the cp genomes of this tribe have a similar structure, gene number and gene arrangement. A phylogenetic tree reconstructed based on 74 protein-coding genes from 70 monocots demonstrates short branch lengths indicating slow evolutionary rates of cp genomes in family Arecaceae

Using in situ seed baiting technique to isolate and identify endophytic and mycorrhizal fungi from seeds of a threatened epiphytic orchid, Dendrobium friedericksianum Rchb.f. (Orchidaceae)

© 2016 Kasetsart University. All orchids require association with mycorrhizal fungi for seed germination and development under natural conditions but their identification and feasibility are not known. The in situ seed baiting germination was examined of Dendrobium friedericksianum Rchb.f., a native threatened epiphytic orchid species, to detect fungi that promote germination. It was found that seed germination percentages were lowered by 0.1%, with a total of seven protocorms formed. Six endophytic fungi were isolated from seven protocorms in seed packets. Three binucleate Rhizoctonia-like fungal isolates which formed a teleomorphic state were morphologically identified as Tulasnella violea, Epulorhiza repen (anamorph Tulasnella) and Trichosporiella multisporum. The species of Beauvaria and Fusarium which are endophyte fungi were also isolated and may play an important role for plant growth and survival of D. friedericksianum. The results in this study suggested that in situ seed baiting is beneficial for screening compatible mycorrhizal fungi to promote the growth and propagation of epiphytic orchids

Towards sex identification of Asian Palmyra palm (Borassus flabellifer L.) by DNA fingerprinting, suppression subtractive hybridization and de novo transcriptome sequencing

Background Asian Palmyra palm, the source of palm-sugar, is dioecious with a long juvenile period requiring at least 12 years to reach its maturity. To date, there is no reliable molecular marker for identifying sexes before the first bloom, limiting crop designs and utilization. We aimed to identify sex-linked markers for this palm using PCR-based DNA fingerprinting, suppression subtractive hybridization (SSH) and transcriptome sequencing. Methods DNA fingerprints were generated between males and females based on RAPD, AFLP, SCoT, modified SCoT, ILP, and SSR techniques. Large-scale cloning and screening of SSH libraries and de novo transcriptome sequencing of male and female cDNA from inflorescences were performed to identify sex-specific genes for developing sex-linked markers. Results Through extensive screening and re-testing of the DNA fingerprints (up to 1,204 primer pairs) and transcripts from SSH (>10,000 clones) and transcriptome data, however, no sex-linked marker was identified. Although de novo transcriptome sequencing of male and female inflorescences provided ∼32 million reads and 187,083 assembled transcripts, PCR analysis of selected sex-highly represented transcripts did not yield any sex-linked marker. This result may suggest the complexity and small sex-determining region of the Asian Palmyra palm. To this end, we provide the first global transcripts of male and female inflorescences of Asian Palmyra palm. Interestingly, sequence annotation revealed a large proportion of transcripts related to sucrose metabolism, which corresponds to the sucrose-rich sap produced in the inflorescences, and these transcripts will be useful for further understanding of sucrose production in sugar crop plants. Provided lists of sex-specific and differential-expressed transcripts would be beneficial to the further study of sexual development and sex-linked markers in palms and related species

Epigenetic changes and transposon reactivation in Thai rice hybrids. Molecular Breeding

Inter- or intraspecific hybridization is the first step in transferring exogenous traits to the germplasm of a recipient crop. One of the complicating factors is the occurrence of epigenetic modifications of the hybrids, which in turn can change their gene expression and phenotype. In this study we present an analysis of epigenome changes in rice hybrids that were obtained by crossing rice cultivars, most of them of indica type and Thai origin. Comparing amplified fragment length polymorphism (AFLP) fingerprints of twenty-four cultivars, we calculated Nei’s indexes for measuring genetic relationships. Epigenetic changes in their hybrids were established using methylation-sensitive AFLP fingerprinting and transposon display of the rice transposable elements (TEs) Stowaway Os-1 and Mashu, leading to the question whether the relationship between parental genomes is a predictor of epigenome changes, TE reactivation and changes in TE methylation. Our study now reveals that the genetic relationship between the parents and DNA methylation changes in their hybrids is not significantly correlated. Moreover, genetic distance correlates only weakly with Mashu reactivation, whereas it does not correlate with Stowaway Os-1 reactivation. Our observations also suggest that epigenome changes in the hybrids are localized events affecting specific chromosomal regions and transposons rather than affecting the genomic methylation landscape as a whole. The weak correlation between genetic distance and Mashu methylation and reactivation points at only limited influence of genetic background on the epigenetic status of the transposon. Our study further demonstrates that hybridizations between and among specific japonica and indica cultivars induce both genomic DNA methylation and reactivation/methylation change in the Stowaway Os-1 and Mashu transposons. The observed epigenetic changes seem to affect the transposons in a clear manner, partly driven by stochastic processes, which may account for a broader phenotypic plasticity of the hybrids. A better understanding of the epigenome changes leading to such transposon activation can lead to the development of novel tools for more variability in future rice breedin

Additional file 1: of Genetic evidence of multiple invasions and a small number of founders of Asian Palmyra palm (Borassus flabellifer) in Thailand

Locations of the B. flabellifer samples in Thailand (DOCX 14 kb