Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

The discovery of thermo-sensitive genic male sterility(TGMS) has led to development of a simple and highly efficient two-line breeding system. In this study, genetic analysis was conducted using three F_2 populations derived from crosses between IR68301 S, an indica TGMS rice line, and IR14632(tropical japonica), Supanburi 91062(indica) and IR67966-188-2-2-1(tropical japonica), respectively.Approximately 1:3 ratio between sterile and normal pollen of F_2 plants from the three populations revealed that TGMS is controlled by a single recessive gene. Bulked segregant analysis using simple sequence repeat(SSR) and insertion-deletion(InDel) markers were used to identify markers linked to the tms gene. The linkage analysis based on the three populations indicated that the tms locus was located on chromosome 2 covering the same area. Using IR68301S × IR14632 F_2 population, the results showed that the tms locus was located between SSR marker RM12676 and InDel marker 2gAP0050058. The genetic distance from the tms gene to these two flanking markers were 1.10 and 0.82 cM, respectively.InDel marker 2gAP004045 located between these two markers showed complete co-segregation with the TGMS phenotype. In addition, InDel marker vf0206114052 showed 2.94 cM linked to the tms gene using F_2 populations of IR68301S × Supanburi 91062. These markers are useful tool for developing new TGMS lines by marker-assisted selection. There were ten genes located between the two flanking markers RM12676 and 2gAP0050058. Using quantitative real-time PCR for expression analysis, 7 of the 10 genes showed expression in panicles, and response to temperatures. These genes could be the candidate gene controlling TGMS in IR68301S.

Mining and validation of novel genotyping-bysequencing(GBS)-based simple sequence repeats(SSRs)and their application for the estimation of the genetic diversity and population structure of coconuts(Cocos nucifera L.)in Thailand

Coconut(Cocos nucifera L.)is an important economic crop in tropical countries.However,the lack of a complete reference genome and the limitations of usable DNA markers hinder genomic studies and the molecular breeding of coconut.Here,we present the results of simple sequence repeat(SSR)mining from a high-throughput genotyping-bysequencing(GBS)study of a collection of 38 coconut accessions.A total of 22,748 SSRs with di-,tri-,tetra-,penta-and hexanucleotide repeats of five or more were identified,2451 of which were defined as polymorphic loci based on locus clustering in 38 coconut accessions,and 315 loci were suitable for the development of SSR markers.One hundred loci were selected,and primer pairs for each SSR locus were designed and validated in 40 coconut accessions.The analysis of 74 polymorphic markers identified between 2 and 9 alleles per locus,with an average of 3.01 alleles.The assessment of the genetic diversity and genetic relationships among the 40 coconut varieties based on the analysis of population structure,principal coordinate analysis(PCoA),and phylogenetic tree analysis using the 74 polymorphic SSR markers revealed three main groups of coconuts in Thailand.The identified SSR loci and SSR markers developed in this study will be useful for the study of coconut diversity and molecular breeding.The SSR mining approach used in this study could be applied to other plant species with a complex genome regardless of the availability of reference genome.

Genetic Diversity and Allelic Frequency of Selected Thai and Exotic Rice Germplasm Using SSR Markers

A collection of 167 Thai and exotic rice accessions was subjected for evaluation of genetic diversity and assessment of relationship by simple sequence repeat (SSR) markers. Among a total of 49 SSR markers, 13 markers distributing over 12 rice chromosomes showed clear polymorphic band patterns, and they were selected for genetic assessment. A total of 110 alleles were detected with an average of 8.46 alleles per locus. The averages of gene diversity, heterozygosity and polymorphic information content were 0.59, 0.02 and 0.56, respectively. The unweighted-pair group method with arithmetic averages (UPGMA) clustering analysis was performed for genetic distance, and phylogenetic tree was constructed. The result showed that this rice collection was divided into two major groups, classified as japonica and indica subspecies. Within the japonica group, temperate japonica and tropical japonica subgroups can be clearly separated. Three-dimensional principal component analysis projection and model-based population structure analysis showed consistent clustering results with two major groups of UPGMA analysis, supporting the classification of japonica and indica subspecies. The indica allelic frequency was also investigated to provide an indicative guide for breeders to overcome the practical problems on sterility of inter-subspecies hybrid offspring. This rice collection and information obtained in this study will be useful for rice breeding programs.

Bartonella species in small mammals and their potential vectors in Asia

In this article,authors review the current knowledge of Bartonella infection in small mammals including rodents,insectivores,bats and exotic small mammal pets and their vectors in Asia.Species of Bartonella are Gram-negative intracellular bacteria that infect erythrocytes of various mammalian and non-mammalian animals and mainly transmitted by blood sucking arthropod vectors.The genus Bartonella includes several species of important human diseases with severe clinical signs.Several new Bartonella species were isolated from rodents and other small mammals,and from human patients in Asia.Bartonella species are identified using standard polymerase chain reaction amplification and a sequencing targeting two housekeeping genes(glt.A and rpoB) and the internal transcribed spacer fragment.Authors also discuss the implications in term of potential emerging zoonotic diseases.

Evaluation of Agronomic Traits in Chromosome Segment Substitution Lines of KDML105 Containing Drought Tolerance QTL under Drought Stress

Drought is a major abiotic constraint to rice production in rainfed lowland and insufficiently irrigated areas. The improvement of drought tolerant varieties is one of the strategies to reduce the negative effects of drought. Quantitative trait loci （QTLs） for primary and secondary traits related to drought tolerance （DT） on chromosomes 1, 3, 4, 8 and 9 that determined from double haploid lines derived from a cross between CT9993 and IR62266 were introgressed and dissected into small pieces in the genetic background of Khao Dawk Mall 105 （KDML105） to develop chromosome segment substitution line （CSSL） population. The CSSLs were evaluated at the reproductive stage for their agronomic performance and yield components under drought stress, and results were compared with irrigated condition. The flowering of CSSL lines was 6 to 7 d earlier than KDML105. The mean values of grain yields in the CSSLs were higher than KDML105 under drought and irrigated conditions. At irrigated condition, the grain yields of introgression lines carrying DT-QTLs from chromosomes 4 and 8 were higher than that of KDML105, whereas other traits showed little difference with KDML105. Analysis indicated that grain yield has positive correlation with plant height, tiller and panicle number per plant, and total grain weight per plant under drought stress while negatively correlated with days to flowering. As mentioned above, CSSLs showing good adaptation under drought stress can be used as genetic materials to improve drought tolerance in Thai rainfed lowland rice breeding program, and as materials to dissect genes underlying drought tolerance.

Ethanol production and mitochondrial-related gene expression of maize(Zea mays) seed during storage

Mitochondrial degradation plays a vital role in seed deterioration.Novel markers were investigated based on a new method for quantifying maize seed deterioration during 12 months’storage under ambient(lab bench,~27℃ and 50-80%relative humidity(RH))or controlled((15±1)℃ and(50±5)%RH in bags with low oxygen permeability)conditions involving two techniques:1)fast ethanol assay and 2)quantitative RT-PCR(qPCR)with four mitochondrial-related genes in maize seed:alcohol dehydrogenase(ADH1),alternative oxidase(AOX1),cytochrome c oxidase(COXc),and ATPase.Ethanol production during imbibition and the expression of genes using the new method were compared to the results of two conventional methods:a germination test and an accelerated ageing test.The results showed that germination following ambient seed storage reduced significantly compared to the controlled conditions,especially at 9 months of storage.Ethanol production of maize seed measured by fast ethanol assay increased during storage.After 6 months,the mean(n=4)ethanol production from seed under ambient conditions was 400μg L^-1 which was higher than under the controlled conditions(240μg L^-1).Stored mRNA level of COXc and ATPase significantly decreased over time in ambient storage but were quite stable under the controlled conditions.Maize seed was also treated for artificial ageing at 42℃ in 100%RH for 12,24,and 48 h.At 24 h after treatment(HAT),maize seed produced significantly more headspace ethanol than at 12 HAT and more than the control(non-treated seed).The transcription level of ADH1 and ethanol production increased.The transcription level of COXc was directly related to the severity of the ageing treatment.In conclusion,a combination of fast ethanol assay and qPCR enhanced understanding of maize seed deterioration and provided new possibilities for the evaluation of seed storability based on transcriptional levels.

A New Approach to Select Doubled Haploid Rice Lines under Salinity Stress Using Indirect Selection Index

This study determined the indirect selection index of doubled haploid(DH)rice using a multivariate analysis approach to select lines adaptive to salinity stress,comprising three experiments.The first experiment involved the selection of good agronomic characters in a field experiment conducted at an experimental station in Bogor,Indonesia.The second experiment involved salinity tolerance screening through hydroponic cultures using 0 and 120 mmol/L NaCl,conducted at a greenhouse in Bogor.The third experiment involved the validation of the indirect adaptability selection index(IASI)through a field experiment in Sukra(saline area).Field experiments followed a randomized complete block design(RCBD),whereas an RCBD nested factorial design was used for the greenhouse experiment.The first and second experiments used 56 DH lines and four check varieties with three replications.In the second experiment,Pokkali and IR29 varieties were also added as tolerant and sensitive checks of salinity,respectively.The third experiment used 28 selected DH lines,Inpari 29 and one sensitive DH line.The good agronomic index(GAI)was 0.465 yield+0.433 number of productive tillers+0.31 number of filled grains.This generated 24 DH rice lines with good agronomic traits.The salinity tolerance index(SaTI)was developed through the average of standardized salinity tolerance score and salinity selection index based on discriminant analysis.This generated 34 DH rice lines with good salinity stress tolerance.The IASI(IASI=GAI–SaTI)selected 28 DH rice lines adaptive to salinity stress and it was considered effective by Sukra validation.

Molecular genetic diversity of winged bean gene pool in Thailand assessed by SSR markers

Winged bean[Psophocarpus tetragonolobus(L.)DC.]is a vegetable legume crop.The center of origin,diversity and domestication of this crop are not known.In this study,we assessed the genetic diversity and population structure of 457 accessions of winged bean collected from six geographical regions(North,Northeast,East,West,and central,and South)in Thailand using 14 simple sequence repeat(SSR)markers.In total,the SSR markers detected only 55 alleles with an average of 3.9 alleles per locus.Observed heterozygosity was relatively high(0.15)and overall gene diversity was moderate(0.487).Gene diversity,allelic richness and observed heterozygosity in the six regions were comparable,while the estimated out-crossing rate was relatively high(16.4%).STRUCTURE analysis grouped the 457 winged bean accessions into three subpopulations.Neighbor-joining(NJ)analysis grouped all the accessions into two major clusters.Genetic groups identified by both STRUCTURE analysis and NJ analysis were unrelated to geographical origins.Principal coordinate analysis revealed no clear clustering of the winged bean accessions.Although genetic groups were not unrelated to geographical origins,most of the winged bean accessions with long pods(30 cm or higher in length)or having purple seed coats or purple young pods were grouped together.This suggested that the winged beans with long pods or with purple seed or purple young pods may have a single origin.Altogether,these results demonstrated that the genetic diversity of winged bean in Thailand was moderate with high genetic admixture.We argue that the high genetic admixture of the winged bean in Thailand is due to seed migration and relatively high outcrossing rate.

Phloem-Mobile AuxlIAA Transcripts Target to the Root Tip and Modify Root Architecture

In plants, the phloem is the component of the vascular system that delivers nutrients and transmits signals from mature leaves to developing sink tissues. Recent studies have identified proteins, mRNA, and small RNA within the phloem sap of several plant species. It is now of considerable interest to elucidate the biological functions of these potential long-distance signal agents, to further our understanding of how plants coordinate their developmental programs at the whole-plant level. In this study, we developed a strategy for the functional analysis of phloem-mobile mRNA by focusing on IAA transcripts, whose mobility has previously been reported in melon （Cucumis melo cv. Hale＇s Best Jumbo）. Indoleacetic acid （IAA） proteins are key transcriptional regulators of auxin signaling, and are involved in a broad range of developmental processes including root development. We used a combination of vasculature-enriched sampling and hetero-grafting techniques to identify IAA18 and IAA28 as phloemmobile transcripts in the model plant Arabidopsis thaliana. Micro-grafting experiments were used to confirm that these IAA transcripts, which are generated in vascular tissues of mature leaves, are then transported into the root system where they negatively regulate lateral root formation. Based on these findings, we present a model in which auxin distribution, in combination with phloem-mobile Aux/IAA transcripts, can determine the sites of auxin action.