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Miniature inverted-repeat transposable elements (MITEs) are numerically predominant transposable elements in the rice genome, and their activities have influenced the evolution of genes. Very little is known about how MITEs can rapidly amplify to thousands in the genome. The rice MITE mPing is quiescent in most cultivars under natural growth conditions, although it is activated by various stresses, such as tissue culture, gamma-ray irradiation, and high hydrostatic pressure. Exceptionally in the temperate japonica rice strain EG4 (cultivar Gimbozu), mPing has reached over 1000 copies in the genome, and is amplifying owing to its active transposition even under natural growth conditions. Being the only active MITE, mPing in EG4 is an appropriate material to study how MITEs amplify in the genome. Here, we provide important findings regarding the transposition and amplification of mPing in EG4. Transposon display of mPing using various tissues of a single EG4 plant revealed that most de novo mPing insertions arise in embryogenesis during the period from 3 to 5 days after pollination (DAP), and a large majority of these insertions are transmissible to the next generation. Locus-specific PCR showed that mPing excisions and insertions arose at the same time (3 to 5 DAP). Moreover, expression analysis and in situ hybridization analysis revealed that Ping, an autonomous partner for mPing, was markedly up-regulated in the 3 DAP embryo of EG4, whereas such up-regulation of Ping was not observed in the mPing-inactive cultivar Nipponbare. These results demonstrate that the early embryogenesis-specific expression of Ping is responsible for the successful amplification of mPing in EG4. This study helps not only to elucidate the whole mechanism of mPing amplification but also to further understand the contribution of MITEs to genome evolution.
Author Summary
Transposable elements are major components of eukaryotic genomes, comprising a large portion of the genome in some species. Miniature inverted-repeat transposable elements (MITEs), which belong to the class II DNA transposable elements, are abundant in gene-rich regions, and their copy numbers are very high; therefore, they have been considered to contribute to genome evolution. Because MITEs are short and have no coding capacity, they cannot transpose their positions without the aid of transposase, provided in trans by their autonomous element(s). It has been unknown how MITEs amplify themselves to high copy numbers in the genome. Our results demonstrate that the rice active MITE mPing is mobilized in the embryo by the developmental stage-specific up-regulation of an autonomous element, Ping, and thereby successfully amplifies itself to a high copy number in the genome. The short-term expression of Ping is thought to be a strategy of the mPing family for amplifying mPing by escaping the silencing mechanism of the host genome.
Citation: Teramoto S, Tsukiyama T, Okumoto Y, Tanisaka T (2014) Early Embryogenesis-Specific Expression of the Rice Transposon Ping Enhances Amplification of the MITE mPing. PLoS Genet 10(6): e1004396. https://doi.org/10.1371/journal.pgen.1004396
Editor: Cédric Feschotte, University of Utah School of Medicine, United States of America
Received: December 25, 2013; Accepted: April 6, 2014; Published: June 12, 2014
Copyright: © 2014 Teramoto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan in the form of Grants-in-Aid for Scientific Research (21380004 and 25292006). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Transposable elements (TEs) are DNA sequences that are capable of jumping from one genomic locus to another and make up a large fraction of eukaryotic genomes. More than 80% of the maize (Zea mays) and barley (Hordeum vulgare) genomes are composed of TEs [1], [2], and they constitute 35% and 14% of the genomes of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), respectively [3], [4]. TEs are harmful to the host because their mobilities perturb genome stability, whereas they play greatly generative roles in genome evolution such as alternation of gene structure, change of expression pattern, and rearrangement of chromosome structure [5], [6].
TEs are classified into two groups according to their transposition mechanisms: class I elements (retrotransposons) that transpose through a copy-and-paste mechanism via an RNA intermediate, and class II elements (transposons) that transpose through a cut-and-paste mechanism without undergoing an RNA intermediate. Class I elements easily attain tens of thousands of copies, whereas the majority of class II elements cannot amplify themselves to 50 copies at most. Unlike other class II elements, miniature inverted-repeat transposable elements (MITEs) have the capacity to amplify themselves to high copy numbers (hundreds or thousands) [7]–[9]. In the rice genome, MITEs are numerically predominant TEs [10], constituting 8.6% of the genome [11]. Because MITEs are too short (<600 bp) to encode any protein, their transpositions must depend on the proteins encoded by the autonomous elements. Well-studied MITEs are classified into the Stowaway and Tourist families, which belong to the Tc1/mariner and PIF/Harbinger superfamilies, respectively. Because MITEs are mainly deployed in gene-rich regions [10], [12] and affect adjacent gene expression [13], they are considered to play an important role in genome evolution. However, little is known about how MITEs attain high copy numbers.
Miniature Ping (mPing) is the first active MITE identified in the rice genome [14]–[16]. Although MITEs are deployed in the genome at a high copy number, the copy number of mPing exceptionally remains at a low level in most rice cultivars: indica and tropical japonica cultivars have fewer than 10 copies, and temperate japonica cultivars including Nipponbare have approximately 50 copies [14]. The transposition of mPing is suppressed in most rice cultivars, but, like other TEs, mPing is activated by exposure to various stress conditions such as gamma-ray irradiation [16], hydrostatic pressurization [17], cell culture [14], anther culture [15], and inhibition of topoisomerase II [18]. Introgression of distantly related genomes also causes mPing transposition [19], [20]. However, mPing is actively transposing without such stresses in the temperate japonica rice strain EG4 (cultivar Gimbozu) under natural growth conditions, and its copy number is approximately 1000 copies [21]. This indicates that mPing has overcome the silencing mechanism or established a novel strategy for its amplification in the EG4 genome. In this sense, mPing in EG4 is an appropriate material to study the amplification of MITEs in plant genomes.
The autonomous element Ping and its distantly related element Pong, which both belong to the PIF/Harbinger superfamily, provide two proteins required for mPing transposition. Both Ping and Pong have two open reading frames (ORFs), ORF1 and ORF2 [22], [23]. The former encodes a Myb-like DNA-binding protein, and the latter encodes a transposase lacking DNA binding domain. Transposase of most class II elements contains a conserved catalytic domain (DDE motif) and a DNA-binding domain [23], [24], whereas these domains are encoded separately by two ORFs in both Ping and Pong [22], [23]. The study of other members of the PIF/Harbinger superfamily suggested that the Myb-like DNA-binding protein directly binds to the subterminal regions of the transposon in order to recruit the transposase [25]. Both Myb-like protein and transposase of either Ping or Pong or both elements are necessary for mPing transposition [22], [23].
In this study, we demonstrate that mPing is actively transposing in the embryo of EG4 during the period from the regionalization of shoot apical meristem (SAM) and radicle to the formation of the first leaf primordium (3 to 5 days after pollination, DAP) with the aid of developmental stage-specific expression of Ping. Our results provide important evidence for the amplification mechanism not only of mPing but also of other MITEs.
Transpositions of mPing during gametogenesis
Plants have acquired the silencing mechanism of TEs in germ cells. In Arabidopsis, for example, TEs are activated specifically in the vegetative nucleus of the pollen, and siRNAs from the activated TEs accumulate in the sperm cells [26]. On the basis of these results, Slotkin and colleagues proposed that siRNAs derived from TEs activated in the vegetative nucleus silence TEs in the sperm cells [26]. We conceived that mPing might overcome such a silencing mechanism in EG4. To confirm this hypothesis, we developed two F1 populations from reciprocal crosses between the mPing-active strain EG4 and the mPing-inactive cultivar Nipponbare, and investigated the transposition activity of mPing by transposon display (TD) analysis. Success of reciprocal crosses was confirmed by PCR analysis using locus-specific primers (Figure S1A). One of the results of TD analysis using two selective bases is shown in Figure 1A; all 16 possible primer combinations were analyzed. The banding patterns of F1 plants were almost the same as those of EG4. The bands that appeared in all F1 plants but not in the parental EG4 plant were derived from another parental Nipponbare plant (Figure S1B). Furthermore, the bands that appeared in only one of eight F1 plants but not in the parental EG4 plant are herein referred as de novo insertions. These bands were confirmed not to be PCR artifacts by sequence and locus-specific PCR analysis (Table S1 and Figure S2). We detected 15.5 de novo insertions per plant in the selfed progenies of EG4, whereas Nipponbare yielded no de novo insertions in the selfed progenies (Figure 1B). This confirmed that mPing is active in EG4 under natural growth conditions but inactive in Nipponbare. If mPing was specifically activated in the pollen of EG4, we could obtain de novo insertions only in the F1 plants from the Nipponbare/EG4 cross. However, we obtained de novo insertions in both Nipponbare/EG4 and EG4/Nipponbare populations (Figure 1B). Moreover, there was no significant difference in the number of de novo insertions per plant between the two F1 populations. This indicates that the activating factor(s) for the mPing transposition is present in both male and female gametes of EG4.
(A) Transposon display (TD) for mPing of the F1 population from reciprocal crosses between EG4 and Nipponbare. One of the results of TD analysis using two selective bases is shown. The cross combinations are indicated at the top of the profiles, respectively. G and F indicate parental EG4 and the F1 plants, respectively. White and black arrowheads indicate the bands representing the de novo mPing insertion and the band derived from Nipponbare genome, respectively. (B) Mean numbers of de novo mPing insertions in a single F1 plant and in a self-pollinated plant. The cross combinations are indicated at the bottom of the profile. All 16 possible primer combinations were analyzed, and mean values were calculated using 16 individuals (n = 16). Bars indicate SE.
Transpositions of mPing during ontogeny of EG4 plants
We performed TD analysis of mPing using genomic DNA samples extracted from endosperm, radicle, and leaf blades of eight progenies (S1) derived from a single parental EG4 plant (S0), and investigated the mPing transposition during ontogeny of rice plants (Figure 2A). One of the results of TD analysis using two selective bases is shown in Figure S3; all 16 possible primer combinations were analyzed. We examined de novo insertions in the same way as described above. Consequently, a total of 228 de novo insertions were detected. These insertions were divided into five groups (Figure 2B): (1) endosperm-specific insertions that appeared only in the endosperm sample, (2) radicle-specific insertions that appeared only in the radicle sample, (3) leaf-specific insertions that appeared only in one sample from the 1st to 3rd leaf blades, but not in the 4th and 5th leaf blades, (4) shoot-specific insertions that appeared in at least one sample of 1st, 2nd, and 3rd leaf blades, and in at least one sample of 4th and 5th leaf blades, and (5) radicle/shoot-specific insertions that appeared in both radicle and leaf blade samples. These de novo insertions were confirmed by sequence and locus-specific PCR analysis (Table S2 and Figure S4). Numbers of each insertion obtained in this study are summarized in Figure 2C. Plant development is divided roughly into three successive phases: embryogenesis, vegetative phase, and reproductive phase. If mPing transposed in the SAM of the S0 plant during vegetative and/or reproductive phases, the de novo insertions would segregate according to Mendel's law among the S1 progenies. We obtained no band that appeared in at least two S1 progenies and was not seen in the S0 plant. This indicates that the transmissible insertion of mPing to the next generation seldom (or never) arises during the vegetative and reproductive phases.
(A) Experimental setup for the ontogenical analysis to determine the timing of mPing transposition in EG4. Eight progenies (S1) derived from a single parental EG4 plant (S0) were grown in a greenhouse. Endosperm, radicle, and leaf blades (1st to 5th) of each S1 plant were sampled for DNA extraction. S2 seeds were harvested from the main culm and the primary tiller of each S1 plant to investigate the inheritance of de novo mPing insertions. The 2nd leaf blade of S0 and S2 plants was also sampled. Detailed information is provided in the 'Materials and Methods'. (B) mPing insertions were detected by transposon display. Representative images of shoot-, leaf-, endosperm-, radicle-, and radicle/shoot-specific insertions are shown. White arrowheads indicate the bands representing the de novo mPing insertion. E: endosperm, R: radicle, L1–L5: 1st to 5th leaf blades. (C) The number of organ-specific de novo insertions in EG4. All 16 possible primer combinations were analyzed. En: endosperm-specific insertion, RS: radicle/shoot-specific insertion, R: radicle-specific insertion, Shoot: shoot-specific insertion, Leaf: leaf-specific insertion. (D) Percentage of leaf positions where the first de novo mPing insertion was found. Shoot: shoot-specific insertion, Leaf: leaf-specific insertion.
Flowering plants have evolved a unique reproductive process called double fertilization. In this process, either of two sperm cells in pollen fuses with either an egg cell or a central cell in the ovule, and then the egg cell fertilized with the sperm cell initiates embryogenesis [27]. In rice, the SAM and radicle are regionalized in the embryo 3 DAP, and three leaves and the radicle are already present in the mature embryo [28]. We detected only three radicle/shoot-specific insertions (Figure 2C), indicating that mPing scarcely transposes during the period from the onset of gametogenesis to the early stage (until 3 DAP) of embryogenesis. Among the 228 de novo insertions, 116 and 17 were shoot-specific and leaf-specific insertions, respectively (Figure 2C). This indicates that mPing actively transposes in the embryo during the period from the regionalization of SAM and radicle (at 3 DAP) to the formation of the 3rd leaf primordia (at 8 DAP). Of the 133 shoot- and leaf-specific insertions, 108 were of the 1st leaf blade (Figure 2D). Since the 1st leaf primordium is formed at 5 DAP, the most active phase of the mPing transposition was considered to be from 3 to 5 DAP. We detected a large number of radicle-specific insertions as well as shoot-specific insertions, and the sum of these insertions accounted for 90% of all insertions detected in this study (Figure 2C). Taken together, we concluded that mPing in EG4 most actively transposes in the 3 to 5 DAP embryo.
Endosperm is a triploid tissue that is produced by fusing a central cell containing two polar nuclei with one of two sperm cells in no particular order. The endosperm formation occurs in parallel with embryogenesis. The endosperm-specific insertions result from the mPing transposition occurred in either gametogenesis or endosperm formation. We observed only two endosperm-specific insertions (Figure 2C), supporting that mPing scarcely transposes during the period from the onset of gametogenesis to the early stage of embryogenesis. The relationship between the banding patterns obtained in TD analysis and the timing of mPing transposition is summarized in Figure S5.
Inheritance of de novo mPing insertions to the next generation
In order for mPing to amplify, the de novo insertions must be transmitted to the next generation. We performed TD analysis using 12 progenies (S2) derived from the main culm and the primary tiller of a single selfed parent (S1) to investigate whether the de novo insertions detected in ontogenical analysis are inheritable (Figure S6). Both radicle-specific and leaf-specific insertions in the S1 plants were not detected in the S2 progenies (0 of 15, 0 of 2, respectively). In contrast, 85% (11 of 13) of the shoot-specific insertions that were detected in the S1 plants also appeared in the S2 progenies. This value (85%) is consistent with the estimated number of inheritable de novo insertions in our previous report [21]. Thus most of the de novo insertions that arose in the 3 to 5 DAP embryo were successfully inherited to the next generation.
Excisions of mPing during ontogeny of EG4 plants
We have already determined the sites of all mPing insertions (1163 in total) in the EG4 genome [13], and have investigated mPing excisions in a small EG4 population using locus-specific primer pairs [29], [30]. Here we examined the timing of the mPing excision with locus-specific PCR using the genomic DNA samples that were used for the ontogenical analysis of the de novo insertion. We randomly chose 48 markers for this study (Table S3). We divided the mPing excisions into five types with the same criteria as those used for the de novo insertions: endosperm-, radicle-, leaf-, shoot-, and radicle/shoot-specific excisions (Figure S7). There were no endosperm-specific and radicle/shoot-specific excisions, indicating that no mPing transposition occurs during the period from the onset of gametogenesis to the early stage of embryogenesis. We detected seven radicle-specific, six leaf-specific, and three shoot-specific excisions. All shoot-specific excisions were detected from the 1st leaf blade sample. These results indicate that, like the de novo insertion, the mPing excision also occurs during the period from the regionalization of the SAM and radicle to the formation of the first leaf primordium, although we cannot completely rule out the possibility that these excisions occur also in somatic cells of mature tissues. Thus, in addition to the experimental results of the de novo insertion, we concluded that mPing of EG4 was most actively transposing in the 3 to 5 DAP embryo.
Expression pattern of Ping in EG4
Both Ping and Pong provide a Myb-like protein and a transposase, which are encoded by their ORF1 and ORF2, respectively (Figure 3A), and have been considered as autonomous elements responsible for the mPing transposition. We investigated the expression of Ping-ORF1, Ping-ORF2, Pong-ORF1, and Pong-ORF2 during embryogenesis to evaluate which autonomous element plays a predominant role in driving the mPing transposition in EG4. Reverse transcription-PCR analysis revealed that Ping-ORF1 and Ping-ORF2 constitutively expressed in the ovary during embryogenesis (Figure 3B). On the other hand, no transcriptions of Pong-ORF1 and Pong-ORF2 (Figure 3B) were observed. This strongly suggests that Ping predominantly controls the mPing transposition in EG4.
(A) Structure of the Ping and Pong elements. Terminal inverted repeats are indicated by black triangles. Boxes represent ORF1 and ORF2, respectively. Gray horizontal arrows indicate the direction of transcription. RNA probes used are indicated below the ORFs. (B) Reverse-transcription PCR analysis of Ping-ORF1, Ping-ORF2, Pong-ORF1, and Pong-ORF2. Numbers in parentheses are PCR cycle numbers. PC: positive control (0.1 ng genomic DNA), NC: negative control (non-reverse-transcribed RNA). (C) Real-time quantitative PCR of Ping-ORF1 and (D) Ping-ORF2. The expression level in the Nipponbare ovary just after pollination was set as 1. The results are presented as means of three biological replicates. Bars indicate SE. (E) The ratio of Ping expression level of EG4 to that of Nipponbare. The means in (C) and (D) were used for calculation.
We performed real-time quantitative PCR (qPCR) analysis to compare the expression level of Ping-ORF1 and -ORF2 between EG4 and Nipponbare during embryogenesis. In all developmental stages from 1 to 6 DAP, the expression levels of both Ping-ORF1 and -ORF2 were higher in EG4 than in Nipponbare (Figure 3C, D). Since EG4 harbors seven copies of Ping, whereas Nipponbare has only one copy (Table S4), the difference in the expression levels between EG4 and Nipponbare is considered to be attributable to the different copy number of Ping. However, we found that Ping of EG4 showed different expression patterns from that of Nipponbare. In Nipponbare, the expression level of Ping-ORF1 and -ORF2 gradually declined until 3 DAP, and restored to the basal level at 6 DAP. In contrast, in EG4, the expression levels of both Ping-ORF1 and -ORF2 rapidly increased, with a peak at 3 DAP (Figure 3C, D). The ratio of relative expression level (EG4/Nipponbare) clearly demonstrated that Ping might be up-regulated in a developmental stage-specific manner in the ovary of EG4 (Figure 3E). Since mPing transposed during the period from 3 to 5 DAP, the rapid increase in Ping expression most likely drive the mPing transposition.
Accumulation of Ping transcripts in the embryo triggers mPing transposition
We investigated the spatial expression pattern of Ping by in situ hybridization using Ping-specific probes. The probe positions were indicated in Figure 3A. The Ping transcripts were detected in all tissues, viz. embryo, endosperm, and ovary wall, in both EG4 and Nipponbare (Figure 4A–C, S8). Among the tissues, the 3 DAP embryo of EG4 yielded an exceptionally strong signal, indicating a high accumulation of Ping transcripts (Figure 4A), whereas the 5 DAP embryo showed a much lower accumulation of Ping transcripts in EG4 (Figure 4D–F). Such a drastic change in accumulation quantity of Ping transcripts with the advance of embryogenesis was consistent with the change in the expression quantity of Ping with the advance of embryogenesis, which was investigated by real-time qPCR (Figure 3C–E). These results suggest that the tissue- and developmental stage-specific accumulation of the Ping transcripts triggers mPing transposition at this stage in EG4. To confirm this hypothesis, we evaluated the spatial expression pattern of Ping in the SAM during the vegetative phase. As described above, mPing hardly transposes in the SAM during this phase. The Ping transcripts were detected in all tissues including the SAM, and, as expected, there was no obvious difference in the signal intensity between EG4 and Nipponbare (Figure 4G–I). Thus the Ping transcripts proved to accumulate developmental stage-specifically only in the tissue where mPing actively transposes. We therefore concluded that the high accumulation of Ping transcripts triggers the transposition of mPing in the 3 DAP embryo of EG4.
Longitudinal sections through the ovary 3 days after pollination of (A, C) EG4 and (B) Nipponbare; the ovary 5 days after pollination of (D, F) EG4 and (E) Nipponbare; and the shoot apical meristem of (G, I) EG4 and (H) Nipponbare seedlings were hybridized with antisense (A, B, D, E, G, H) or sense (C, F, I) RNA probes. Little staining was obtained with the sense probe (F). Em: embryo, En: endosperm, OW: ovary wall, SAM: shoot apical meristem. Scale bars represent 100 µm.
SNP in an intronic region of Ping-ORF1
EG4 has seven Ping elements (Ping-1 to -7), whereas Nipponbare has only one (Ping-N) (Table S4). When we sequenced and compared all Ping elements, a single nucleotide polymorphism (SNP) in the first intronic region of Ping-ORF1 was detected between EG4 and Nipponbare (Figure 5A). Ping-N has a 'T' nucleotide on the SNP region, whereas all Ping elements in EG4 have a 'C' nucleotide. We named the former 'T-type Ping' and the latter 'C-type Ping'.
(A) Determination of the SNP sequence in the first intronic region of Ping-ORF1. The arrowhead indicates the position of the SNP. The number indicates the position of the Ping element. Ping harboring +1261C SNP and +1261T are named 'C-type' and 'T-type' Ping, respectively. (B) Box plots of mPing copy number in AG lines. The top and bottom of the boxes mark the first and third quartiles, respectively. The center line represents the median, and the whiskers show the range of observed values within 1.5 times the interquartile range from the hinges. Values beyond 1.5 times the interquartile range from the nearest hinge are marked by open circles. 'No Ping', 'C-type Ping,' and 'T-type Ping' indicate the groups having no Ping, C-type Ping, and T-type Ping, respectively. Expression of (C) Ping-ORF1 and (D) Ping-ORF2 during embryogenesis in mPing-active strains (A119 and A123) and mPing-inactive strains (A105 and G190). The results are presented as means of three biological replicates. Bars indicate SE. The ratio of (E) Ping-ORF1 and (F) -ORF2 expression level of A105, A119, A123, and G190 to that of Nipponbare. The means in (Fig. 3C) and (Fig. 3D) were used for calculation.
In addition to EG4, several Aikoku and Gimbozu landraces (hereafter AG strains) are known to exhibit high mPing activity [21]. We investigated the SNP-type of Ping and the copy number of Ping and mPing in 93 AG strains, and evaluated the effect of C-type Ping on the mPing activity. These 93 AG strains were divided into three groups according to the SNP-type of the Ping allele (Table S4): strains harboring C-type Ping; strains harboring T-type Ping; and strains harboring no Ping. The strains with C-type Ping had more mPing copies than those with T-type Ping or no Ping (Figure 5B, Steel–Dwass test, p<0.01). This implies that the C-type Ping could drive the mPing transposition. We further investigated the expression patterns of Ping-ORF1 and -ORF2 in two mPing-active strains (A119 and A123) and two mPing-inactive strains (A105 and G190) during embryogenesis (from 1 to 6 DAP). A119 and A123 have six and ten copies of C-type Ping, respectively, and both A105 and G190 have one copy of T-type Ping (Table S4). Expression analysis revealed that A105 and G190 kept low expression levels of Ping-ORF1 and -ORF2, whereas A119 and A123 showed high expression levels with a peak around 3 DAP (Figure 5C–F). This indicates that, in EG4, A119, and A123, the developmental stage-specific expression of Ping is controlled by the same factor(s) described in the Discussion.
Our final goal was to elucidate how MITEs attain their high copy numbers in the genome. To this end, we chose mPing, which is the only active MITE identified in rice, as a material and analyzed the timing of mPing transposition in the mPing-active strain EG4. Consequently, we successfully found one mechanism of the mPing amplification; mPing most actively transposes during the period from the regionalization of the SAM and radicle to the formation of the first leaf primordium (3 to 5 DAP) by the developmental stage-specific up-regulation of the autonomous element Ping.
The transpositions of TEs are categorized into germinal and somatic types according to the type of cells where the transposition takes place. LORE1a in Lotus japonicus is activated in plants regenerated from de-differentiated cells and transposes in male germ cells by the pollen grain-specific LORE1a transcription, resulting in the asymmetric transposition of LORE1a in the reciprocal crosses between the active and non-active lines [31]. Tag1 in Arabidopsis shows germinal transposition activity in both male and female germ cells. Consequently, the reciprocal crosses show symmetric transposition of Tag1 [32]. These results demonstrate that the transposition activity in reciprocal crosses reflects the tissue specificity of germinal transposition. In this study, reciprocal crosses between EG4 and Nipponbare showed the same mPing transposition activity, which may suggest that mPing in EG4 transposes in both male and female germ cells. However, we obtained only a few de novo endosperm-specific and radicle/shoot-specific insertions in the ontogenical analysis, although we detected a number of de novo shoot-specific and radicle-specific insertions. We therefore concluded that most mPing transposes not in germ cells but in somatic cells after pollination. Somatic transposition that occurs at the late stage of plant development often produces spotted and striped segments in tissues, such as maize seed coat variegation caused by Mutator excision from the bz2 gene [33], [34] and rice leaf color variegation by nDart1excision from the OsClpP5 gene [35]. In animals, somatic transposition is seldom transmitted to the next generation because germ cells are set aside from somatic cells at the early stage of embryogenesis. On the other hand, in plants, germ cells are generated from somatic cells at the reproductive stage. In rice, gametes are generated in the SAM; therefore, somatic transposition that occurred in the SAM can be transmitted to the next generation via gametes. In this study, we revealed that most mPing elements transposed in somatic cells of the embryo during the period from 3 to 5 DAP. Being a class II TE that transposes by a cut-and-paste mechanism, mPing is expected to be eliminated from genomic DNA with a certain frequency. However, a previous report demonstrated that the mPing excision sites would be repaired by utilizing a copy of mPing from either the sister chromatid or from the homologous chromosome [29]. The mPing excision site cannot be repaired if mPing transposes in germ cells, which are haploid. It is therefore considered that the somatic transposition of mPing is an important factor for mPing amplification in the genome.
The autonomous elements Ping and Pong mediate mPing transposition in the rice genome. Many japonica cultivars, including EG4 and Nipponbare, have both Ping and Pong. This study demonstrated that Ping plays a predominant role in mPing transposition in EG4. However, a heterologous expression assay using Arabidopsis and yeast showed that Pong had a higher catalytic capacity for mPing transposition than Ping [22], [23]. Furthermore, mPing transposition was observed under stress conditions in several rice cultivars harboring only Pong [14], [17], [19]. In this study, however, we detected very low expression of Pong through the development of rice plants, indicating that Pong would be epigenetically silenced at the transcriptional level in EG4. In contrast, Ping constitutively expressed in all organs including the SAM and embryo. Nevertheless, mPing could be transposing most actively in the embryo during the period from 3 to 5 DAP. Since the stage-specific up-regulation of Ping was observed during the period of mPing transposition, we hypothesized that the expression level of Ping needed to exceed a certain threshold of mPing transposition.
All mPing-active strains (EG4, A119, and A123) showed higher expression of Ping with a peak around 3 DAP than the mPing-inactive strains (Nipponbare, A105, and G190). Although further experiments are needed to elucidate the mechanism of developmental stage-specific up-regulation of Ping expression, we propose two hypotheses: (1) position- and dosage-effect, and (2) effect of SNP. The details of the hypotheses are as follows.
Position- and dosage-effect
Chromosomal position and copy number of TE often affect the transposition activity. The former is known as 'position effect' and the latter as 'dosage effect'. Eight independent Tam3 copies residing in the Antirrhinum majus genome show different transposition activities from each other [36]. In Arabidopsis, germinal reversion frequency of Tag1 increases in proportion to its copy number [32]. The mPing-inactive strains Nipponbare, A105, and G190 have only one Ping at the same locus, whereas the mPing-active strains EG4, A119, and A123 have respectively seven, six, and ten copies of Ping at different loci except for the Ping-1 locus. Furthermore, the expression pattern of Ping showed slight variation among the mPing-active strains harboring only C-type Ping. These results suggest that the developmental stage-specific up-regulation of Ping expression is probably regulated by the position-effect and/or the dosage-effect.
Effect of SNP
Intronic SNPs are known to cause drastic effects on gene expression. In humans, an intronic SNP in SLC22A4 affects transcriptional efficiency in vitro, owing to an allelic difference in affinity to the transcriptional factor RUNX1 [37]. Furthermore, a SNP located in the intronic enhancer region of the thyroid hormone receptor β gene enhances pituitary cell-specific transcriptional activity [38]. In this study, we demonstrated that a SNP is present in the intronic region of Ping-ORF1, and Ping elements in the AG strains were categorized into either T-type or C-type Ping according to the SNP-type. Since all strains that showed a peak in the expression analysis had only C-type Ping, the intronic SNP might influence the developmental stage-specific up-regulation of Ping expression. T-type Ping was present in 14 AG strains as one copy, and its chromosomal location did not differ among strains. In contrast, the copy number of C-type Ping varied from one to ten, and their chromosomal locations, except for Ping-1, differed from each other. These results indicate that T-type Ping has lost its activity, whereas C-type Ping may be still active in the rice genome. Furthermore, we found that the copy number of mPing was significantly larger in strains harboring C-type Ping than in strains harboring T-type Ping. This strongly supports that C-type SNPs in the intronic region of Ping contribute to the amplification of mPing, presumably by the developmental stage-specific up-regulation of Ping expression.
Since the transposition of TEs often damages the host genome, TEs with high transposition activity are targeted by the silencing mechanisms. Nevertheless, MITEs amplify to very high copy numbers not only in plant genomes but also in animal genomes. Very little is known about how MITEs attain their high copy numbers by escaping the silencing mechanism. The transposition of mPing is transiently induced by various stresses [14]–[18], indicating that the activity of mPing is suppressed by the silencing mechanisms in many cultivars. Thus, mPing must overcome the silencing mechanism in order to maintain the transposition activity under natural growth conditions. Our results revealed that mPing in EG4 was mobilized by the sufficient supply of Ping transcripts produced only during the period of mPing transposition. This stage-specific activation is thought to be a strategy of the mPing family to amplify mPing by escaping from the silencing mechanism of the host genome. Since no active MITEs other than mPing so far have been identified, it is very difficult to elucidate if the other MITEs also attain their high copy numbers in the same way as mPing amplifies. Given that the other active MITEs are identified, however, our study will help to understand their amplification mechanisms. Our previous study documented the generation of new regulatory networks by a subset of mPing insertions that render adjacent genes stress inducible [13]. In addition to mPing, other MITEs also contribute to gene and genome evolution via providing new promoter regulatory sequences, transcriptional termination elements, and new alternative exons [39], suggesting that the amplification of MITEs causes gene and genome evolution. Our results provide clues to further understand not only the amplification mechanism of MITEs but also the co-evolution of MITEs and the host genome.
Materials and Methods
Plant materials and sampling
EG4 (cultivar Gimbozu), Nipponbare, and 94 Aikoku/Gimbozu landraces were used in this study (Table S4). Aikoku/Gimbozu landraces were provided from the GenBank project of the National Institute of Agrobiological Science, Ibaraki, Japan. Reciprocal crosses between EG4 and Nipponbare were made in a green house. Before pollination, all anthers were removed from the flowers of maternal plants. The pollinated flowers were covered with protective bags to prevent outcrossing until harvest. After harvesting, success of crosses was checked with the molecular markers. For ontogenical analysis, eight progenies of EG4 (S1) derived from a single parental plant (S0) were grown in a greenhouse, and all S2 seeds were harvested. For S1 plants, each seed was cut into two halves, and the half including the embryo was germinated and the other was sampled. After germination, the radicle and the 1st, 2nd, 3rd, 4th, and 5th leaf blades were sampled. The second leaf was collected from S0 and S2 plants. For estimation of Ping and mPing copy numbers, eight bulked plants were sampled. For RNA extraction, ovaries before pollination and ovaries at 1, 2, 3, 4, 5, and 6 DAP were collected. All samples were immediately frozen in liquid nitrogen and stored at −80°C until use.
DNA extraction and transposon display
DNA extraction and transposon display was performed following a published protocol [30]. For DNA extraction from endosperm, we used GM quicker 2 (Nippon Gene).
Locus-specific PCR
Sequencing of mPing-flanking fragments excised from transposon display gels and primer design were performed following a published protocol [30]. The genomic locations of the mPing insertion sites were forecasted by a BLAST search in the Rice Annotation Project Database (RAP-DB; http://rapdb.dna.affrc.go.jp/) [40], [41] using mPing flanking sequences as queries. To prepare enough templates for PCR, whole genome amplification was performed using an illustra GenomiPhi V2 Kit (GE Healthcare). mPing excision was detected by PCR with mPing-sequence characterized amplified region (SCAR) markers [29]. PCR was performed in 10-µl reaction volumes containing 10 ng of the template DNA, 5 µl of GoTaq Green Master mix (Promega), 5% DMSO, and 0.25 µM of each primer (Table S3). PCR conditions were as follows: 94°C for 3 min; 40 cycles of 98°C for 10 s, 57°C for 30 s, and 72°C for 45 s; and 72°C for 5 min. To detect the presence of Ping-N, -1, -2, -3, -4, -5, -6, and -7, eight Ping-SCAR markers were used. The genomic locations of the Ping insertion sites were referred from a previous report [42]. For detection of the Ping-1 allele, PCR was performed in 10-µl reaction volumes containing 10 ng of template DNA, 0.2 U of KOD FX Neo (Toyobo), 1×PCR buffer for KOD FX Neo (Toyobo), and 0.2 µM of each primer (Table S5). PCR conditions were as follows: 94°C for 3 min; 35 cycles of 98°C for 10 s, 60°C for 30 s, and 68°C for 90 s; and 72°C for 1 min. For detection of Ping-N, -2, -3, -4, -5, -6, and -7 alleles, PCR was performed in 10-µl reaction volumes containing 10 ng of template DNA, 5 µl of GoTaq Green Master mix (Promega), 5% DMSO, and 0.25 µM of each primer (Table S5). PCR conditions were as follows: 94°C for 3 min; 35 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 45 s; and 72°C for 1 min.
RNA isolation and expression analysis
Total RNA was isolated using TriPure isolation reagent (Roche) and digested using RNase-free DNase (TaKaRa). First strand cDNA was synthesized using a Transcriptor first strand cDNA synthesis kit (Roche). For reverse transcription PCR, PCR was performed in 10 µl reaction volumes containing cDNA generated from 4 ng total RNA, 0.2 U of KOD FX Neo (Toyobo), 1×PCR buffer for KOD FX Neo (Toyobo), and 0.5 µM of each primer. PCR conditions were as follows: 94°C for 3 min; 35 or 45 cycles of 98°C for 10 s, 60°C for 10 s, and 68°C for 10 s. Relative quantification of Ping-ORF1 and Ping-ORF2 were calculated by the 2−ΔΔCT method [43] using Light cycler 1.5 (Roche). The UBQ5 gene was used as the calibrator gene. The thermal profile consisted of 10 min at 95°C; and 45 cycles of 4 s at 95°C, 10 s at 60°C, and 1 s at 72°C. Amplification data were collected at the end of each extension step. The primer pairs used in this study are listed in Table S6.
Paraffin sectioning and in situ hybridization
Plant samples were fixed with 4% (w/v) paraformaldehyde and 1% Triton X in 0.1M sodium phosphate buffer for 48 h at 4°C. They were then dehydrated in a graded ethanol series, substituted with 1-butanol, and embedded in Paraplast Plus. The samples were sectioned at 8-µm thickness using a rotary microtome. Fragments of Ping-ORF1 (1091 bp) and Ping-ORF2 (1368 bp) were cloned into pBlueScript SK+ (Stratagene) and sequenced. For digoxigenin-labeled antisense/sense RNA probe synthesis, in vitro transcription was performed using T7 RNA polymerase and T3 RNA polymerase. In situ hybridization and immunological detection with alkaline phosphatase were performed according to Kouchi and Hata [44].
SNP detection
PCR was performed in 10-µl reaction volumes containing 10 ng of template of DNA, 5 µl of GoTaq Green Master mix (Promega), 5% DMSO, and 0.25 µM of each primer. PCR conditions were as follows: 94°C for 3 min; 35 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 30 s; and 72°C for 1 min. PCR primers used in this study are listed in Table S6. Because the original sequence contained an Afa I restriction site, one mutation was introduced into the reverse primer. The 5-µl PCR products were mixed with 5 µl restriction mixture containing 1 U Afa I (TaKaRa), 33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 0.5 mM Dithiothreitol, 66 mM K-acetate, and 0.01% (w/v) bovine serum albumin. After 16 h incubation at 37°C, DNA gel electrophoresis was performed. PCR products (502 bp) including +1261T SNP were not digested, whereas PCR products including +1261C SNP were digested into two fragments (352 bp and 150 bp).
Estimation of Ping and mPing copy number
To determine the copy number of Ping by Southern blot analysis, genomic DNA samples were digested with Eco RI restriction enzyme. These samples were loaded onto an agarose gel, separated by electrophoresis, blotted onto a nylon membrane, and probed with the Ping fragment. The mPing copy number was determined by real-time quantitative PCR as described previously [45] with little modification. Quantitative PCR was performed using the LightCycler 480 system (Roche). PCR was performed in 20 µl reaction volumes containing 5 µl genomic DNA (0.4 ng/µl), 1×LightCycler 480 SYBR Green I Master mix (Roche), and 0.5 µM of each primer. Specificity of the amplified PCR product was assessed by performing a melting curve analysis on the LightCycler 480 system.
Supporting Information
Figure S1.
Verification of the reciprocal crosses between EG4 and Nipponbare. (A) Locus-specific PCR analysis of Ping with locus-specific markers. The maker names and cross combinations are indicated at the top of the profiles. (B) Locus-specific PCR analysis of mPing in Nipponbare genome. The genomic location of the mPing insertions and cross combinations are indicated at the top of the profiles. G and F indicate parental EG4 plants and the F1 plants, respectively. Lane M: DNA size marker (Gene Ladder 100, Nippon Gene), Lane N: Nipponbare. Black and white arrowheads show the bands indicating the presence and absence of Ping/mPing, respectively.
Figure S2.
Locus-specific PCR analysis of de novo mPing insertions in F1 progenies. Ten representative results are shown. The genomic locations of the mPing insertions and cross combinations are indicated at the top of the profiles. G and F indicate parental EG4 and the F1 plants, respectively. Lane M: DNA size marker (Gene Ladder 100, Nippon Gene), Lane N: Nipponbare. Black and white arrowheads show the bands indicating the presence and absence of mPing, respectively.
Figure S3.
Ontogenical analysis of mPing transposition in EG4 by transposon display. Eight progenies (S1) were derived from a single parental EG4 plant (S0). The 2nd leaf blade of the S0 plant and the endosperm, radicle, and leaf blades of each S1 plant were sampled and subjected to transposon display. White, red, and green arrowheads indicate shoot-, radicle-, and leaf-specific insertions, respectively. The rice plant has alternate distichous leaves; therefore, we analyzed the insertion in both [n+1]th and [n+2]th leaves to confirm whether the insertion detected in [n]th leaf is leaf-specific or shoot-specific. But we did not investigate the specificity of the insertions detected in the 4th and 5th leaves using their upper leaves. For this reason, we did not categorize such insertions and marked with the gray arrowhead. E: endosperm; R: radicle; L1–L5: 1st to 5th leaf. For progeny 2–8, samples are applied in the same order as for progeny 1.
Figure S4.
Locus-specific PCR analysis of de novo mPing insertions in various tissues of a single EG4 plant. Eight representative results are shown. The genomic locations of the mPing insertions and insertion types are indicated at the top of the profiles. Lane M: DNA size marker (Gene Ladder 100, Nippon Gene), Lane N: Nipponbare, Lane E: endosperm, Lane R: radicle, Lane L1–L5: 1st to 5th leaf. Black and white arrowheads show the bands indicating the presence and absence of mPing, respectively.
Figure S5.
Schematic representation of the relationship between banding patterns obtained in transposon display and the timing of mPing transposition. If mPing transposes in the period indicated by the red bar, the schematic banding patterns indicated by the arrows will be obtained. E: endosperm, R: radicle, L1–L5: 1st to 5th leaf blade, DAP: days after pollination, SAM: shoot apical meristem.
Figure S6.
Inheritance of de novo mPing insertions in EG4. S2 plants derived from the main culm and the primary tiller of a single S1 plant were assayed. The shoot-specific insertion in the S1 plant (white arrowhead) was inherited by S2 plants, whereas the radicle-specific insertion (red arrowhead) was not. E: endosperm; R: radicle; L1–L5: 1st to 5th leaf.
Figure S7.
mPing excisions in EG4. mPing excisions were detected by locus-specific PCR using the genomic DNA samples that were used for the ontogenical analysis of the de novo insertion. We analyzed 48 loci. Black and white arrowheads show the bands indicating the presence and absence of mPing, respectively. Figures indicate (A, B) shoot-specific excisions, (C, D) leaf-specific excisions, and (E) radicle-specific excision. G: EG4 (S0 plant); N: Nipponbare; E: endosperm; R: radicle; L1–L5: 1st to 5th leaf.
Figure S8.
Detection of Ping-ORF2 spatial expression patterns by in situ hybridization analysis. Longitudinal sections through the ovary 3 days after pollination of (A) EG4 and (B, C) Nipponbare were hybridized with (A, B) antisense or (C) sense RNA probes.
Table S1.
De novo mPing insertion sites detected in F1 progenies.
Table S2.
De novo mPing insertion sites detected in various tissues of a single EG4 plant.
Table S3.
mPing-SCAR markers used in this study.
Table S4.
Plant materials used in this study.
Table S5.
Ping-SCAR markers used in this study.
Table S6.
Primer pairs used in this study.
Author Contributions
Conceived and designed the experiments: TTs. Performed the experiments: ST. Analyzed the data: ST. Contributed reagents/materials/analysis tools: YO TTa. Wrote the paper: ST TTs.
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Start by thinking about your audience. What kinds of products or services are they most interested in? Which products or services are you currently using that you would feel comfortable recommending to your readers? You can add links to any affiliate program to your site, but if you want to market them with authenticity, it's best to stick to companies with which you already have some kind of connection.
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Robert said he did an average of 4-6 of these gigs per year for a while depending on his schedule and the work involved. The best part is, he charged a flat rate that usually worked out to around $100 per hour. And remember, this was pay he was earning to advise people on the best ways to use social media tools like Facebook and Pinterest to grow their brands.
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Merchants receiving a large percentage of their revenue from the affiliate channel can become reliant on their affiliate partners. This can lead to affiliate marketers leveraging their important status to receive higher commissions and better deals with their advertisers. Whether it's CPA, CPL, or CPC commission structures, there are a lot of high paying affiliate programs and affiliate marketers are in the driver's seat.
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The terms of an affiliate marketing program are set by the company wanting to advertise. Early on, companies were largely paying cost per click (traffic) or cost per mile (impressions) on banner advertisements. As the technology evolved, the focus turned to commissions on actual sales or qualified leads. The early affiliate marketing programs were vulnerable to fraud because clicks could be generated by software, as could impressions.
While I was doing WordPress speed optimization I noticed lots of people needed it, but very few people supplied it (there were a lack of services and tutorials when I researched Google). I also knew hosting was the #1 factor of website speed factor and these companies paid up to $200/sale. Hosting is a competitive space but the commissions and lack of supply enticed me.


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Error 2753 Accrdsub.exe
that belongs to ActivClient, a desktop authentication software that works with smart cards, readers, operating systems, networks and enterprise applications. This is not a critical Windows component and should be removed if known to cause problems. ActivClient software enables the enforcement of company guidelines and policies while accessing networks and workstations. The program features storage for multiple credentials using the public key infrastructure (PKI); one-time password (OTP) and static passwords on a single authentication device; remote access solutions; PIN management; notifications and logging. ActivIdentity is an American company based in Silicon Valley, California that develops digital identity assurance solutions including authentication and smart card services for enterprise, government and commercial establishments. The company was founded in 1987 and acquired by HID Global in 2010. Accrdsub stands for ActivIdentity card event subhandler The .exe extension on a filename indicates an executable file. Executable files may, in some cases, harm your computer. Therefore, please read below to decide for yourself whether the accrdsub.exe on your computer is a Trojan that you should remove, or whether it is a file belonging to the Windows operating system or to a trusted application. Click to Run a Free Scan for accrdsub.exe related errors Accrdsub.exe file information Accrdsub.exe process in Windows TaskManager The process known as ActivIdentity card event handler belongs to software ActivClient (version 6.1 x86, x86) or ActivClient CAC (version 6.1 x86, 6.1 AFR) or ActivClient for CAC - PKI Only or ActivClient Mini or SAMSUNG USB Driver for Mobile by ActivIdentity (www.actividentity.com). Description: Accrdsub.exe is not essential for the Windows OS and causes relatively few problems. The file
be down. Please try the request again. Your cache administrator is webmaster. Generated Tue, 11 Oct 2016 04:16:14 GMT by s_ac15 (squid/3.5.20)
Installer (MSI) Windows Installer (MSI) Error Messages NORTEL NETWORKS Java Device Manager 6 One of the user's experiences an error which i am not able to replicate during http://www.itninja.com/question/installer-error-2753 testing. I have a CA to set permissions using cacls.exe. However one user generating a 2753 error on calcs.exe. 2753 The File '[2]' is not marked for installation. Fixed the problem https://forums.indigorose.com/forum/msi-factory-2-1/msi-factory-2-discussion/29962-error-2753-the-file-filename-exe-is-not-marked-for-installation by changing the permissions manually. Just wanted to know what could trigger this error? Thank you. 0 Comments [ + ] Show Comments Comments Please log in to comment Rating comments error 2753 in this legacy AppDeploy message board thread won't reorder them,so that the conversation will remain readable. Answers 0 I would duplicate the problem and during the procedure have PROCMON running to capture the error, this way you will get a more accurate look at what is going on. Answered 11/30/2007 by: Coriolus Please log in to comment Please log in to comment 0 will error 2753 accrdsub.exe the windows cleanup utility be able to resolve this issue? will tha patch be able to delete the previous windows install Answered 07/15/2008 by: jonaman_17 Please log in to comment Please log in to comment 0 ORIGINAL: jonaman_17 will the windows cleanup utility be able to resolve this issue? will tha patch be able to delete the previous windows install If you had used the tools available here on AppDeploy, in this case the 'MSI Error's look-up page http://itninja.com/question/help-with-msi-132779,you would see that the error is connected with the installation packge, not Windows, so any clean-up exercise would be pointless. Answered 07/16/2008 by: VBScab Please log in to comment Please log in to comment 0 Got error 2753 the file 'swdnld.exe' is not marked for installation when trying to manually install shockwave player version 11.5.9. I renamed the file swdnld.exe to swdnld.exe.old then rebooted and reinstalled Shockwave.msi Player w/o issues. Answered 01/04/2011 by: ihudson Please log in to comment Please log in to comment 0 Issue:: Error 2753 the file 'xxx.exe' is not marked for installation Scenario:: Your machine has application X version 1.0 (x_1.0)already installed and you are trying
Factory 2.2 MSI Factory 2 Discussion Welcome to the Indigo Rose Software discussion forums! You will need to sign up for a forum account and login before you can post. Need help? View the Forum FAQ. Announcement Collapse No announcement yet. Error 2753: The File 'filename.exe' is not marked for installation Collapse X Collapse Posts Latest Activity Search Page of 1 Filter Time All Time Today Last Week Last Month Show All Discussions only Photos only Videos only Links only Polls only Filtered by: Clear All new posts Leo Vildosola Forum Member Join Date: May 2010 Posts: 15 #1 Error 2753: The File 'filename.exe' is not marked for installation 05-01-2010, 10:52 AM I am trying to run a custom action on a conditionally installed component and I get the following error. Error 2753: The File 'filename.exe' is not marked for installation. What I don't understand is that the component is installed correctly (that is, the condition evaluates to TRUE). However, the action fails. I use the following settings: Type: Run Executable File ID: filename.exe Scheduling: Immediate Return: Ignore Furthermore, I tried using other permutations of Run Executable but it does not work. BTW, I used this same technique before but this is the first time where a Condition other than 1 is used. Tags: 2753, custom action, error, run executable Leo Vildosola Forum Member Join Date: May 2010 Posts: 15 #2 05-02-2010, 05:18 AM I solved my own problem using a LuaScript instead and used the Run LuaScript custom action. Here's the code I used in RunExe.lua. Code: local runExe = MSI.GetProperty(_hInstall, "RUN_EXE"); if (runExe) then exe = MSI.GetTargetPath(_hInstall, "INSTALLDIR") .. "filename.exe"; Shell.Execute(exe); end Where RUN_EXE is an integer property bound to a checkbox control defined under Project Settings and configured to Allow transfer to server side (property is secure). Comment Post Cancel Previous Next Help AutoPlay Media Studio Setup Factory TrueUpdate Visual Patch Go to top Powered by vBulletin Version 5.2.3 Copyright © 2016 vBulletin Solutions, Inc. All rights reserved. Copyright © 2016 Indigo Rose Software. All rights reserved. All times are GMT-6. This page was generated at 1 minute ago. Working... Yes No OK OK Cancel X


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Chai's history, a mystery?
Now that you've met our Founder, Gaurav and heard a bit about his story, we want say, "Hello world!" with our first blog post by reflecting on the origins of authentic Indian chai. How did this delicious drink begin? Who had the idea to bring together black tea, milk and spices?
In Indian Sub Continent, where ever you look, you will see that chai crosses all socio-income classes, all communities, all people. Chai is a unifying cultural artifact and a soothing peacemaker.
Chai is the go-to drink in the morning, noon and evening. It's a social drink, a communal drink, an addictive drink, a tradition. It's the drink you make before sitting down to write your midterm paper, before having an important meeting, before a delicious meal (ok sometimes after too). It's a drink you make when you have friends over and for family gatherings. It's a drink you have on important days, but also on regular days. It's the welcoming drink with friends and family and offering of chai often leads to people sitting down, longer than planned to share stories, gossip, pontificate, and dream.
It's a true ritual in our lives, it often is the start of something great, big or small and we love it.
As we reflect on this ritual, we remember the stories, the folklore, the myths of how chai came to be. We've mostly heard quick answers about the origins of chai from our parents — "Chai came from our grandparents who said they were given a recipe from their parents, who were given a recipe from their parents"…and so on.
From generations past, each family proudly brews their own home-made recipe. Some people like a mix of cardamom and ginger, others include cinnamon and star anise, can't forget a blend of spices and sugar. Everyone has their own unique blend. We honor that and remember our grandparents, great grandparents and beyond who have verbally passed down these delicious, addictive recipes to us.
But how far back can we trace chai, beyond a few generations of our ancestors? Chai simply translates to "tea" but to those in the Indian Subcontinent it implies tea, fused with milk, sugar and spices. Keeping this in mind, we look far back to the first references of chai (tea) and found a few starting points.
Around 3000 BC: Ayurvedic Ritual in Ancient India
Let's teleport our imagination as far back as 5000 years ago. It is said, that in a fancy royal court in India a King was found brewing healing potions. It's possible, chai (different from the current, known version) made it's debut here. This King wanted to make an all-round, Ayurvedic drink for his people.
He carefully selected each ingredient to help with healing the body and based his selection on commonly known benefits as defined in classic Ayurveda:
- Ginger and black pepper: stimulate digestion
- Cloves: antiseptic properties to relieve pain
- Cardamom: elevate mood
- Cinnamon: stimulate circulation and respiration
- Star anise: to freshen breath
Back in the day, chai was a concoction of these, and possibly other, delicious spices and herbs, served as a warm drink: no milk or tea was involved. While simply different from what we know today, chai was still a drink of significance, meaning and purpose.Around 1500 to 500 BC: Vedic Period Myths
There is a lore told verbally that we found amongst our families. This story dates to a story from Hindu mythology. We found our families interchanged the reference to Sanjeevani, a healing herb, with that of the concoction of chai!
It might be a bit of a stretch to imagine the story was referring a cup of chai, nonetheless, this is a beautiful, meaningful story.
Around 1200 BC: Indigenous Tribal Ritual
There is another reference to chai, in the 12th century with the Singpho tribe, an indigenous tribe that habited India, China and Myanmar. The tribes in Arunachal Pradesh and Assam are known to have be the first drinkers of tea, or chai.
The Singpho people produced tea by plucking leaves of Assam which they dried and smoked to then steep and enjoyed as a warm drink. The Singhpo people are said to have given the British the idea of making tea many centuries later.
"Steam rises from a cup of tea and we are wrapped in history, inhaling ancient times and lands, comfort of ages in our hands" — Faith Greenbowl
Around the 1800's: The British inspiration
The British, ruling India at the time, began to cultivate tea for themselves in regions of Assam after connecting with and being inspired by the Singpho tribe's practice. Up until that point, they relied on China for their tea, and this discovery gave them direct access to something they already loved and embraced in their culture. For the Brit's, tea time simply meant black tea with milk.
After satisfying the needs for tea in England, with the same tea found in India, the British sought to increase the consumer market and introduced tea, as a product, in India. They instituted mandatory tea breaks at work, starting with Indian railways. Many Tea vendors, "Chai Wallahs", started propping on railway lines, who started fusing their own spices with milk and sugar, much like all other South Asian fare, giving rise to the authentic chai we find today — full of aromatic flavors and freshness.
Soon, from the streets, simmering pots were found inside homes, brewed frequently with pride and shared widely with generosity. A new cultural icon of South Asian culture was truly in full form.
Reflecting: An infusion with deep meaning and purpose
In the end, we may not know exactly when the first pot of chai was brewed, by who, and why. But stories like this give us some view into the depth, texture and inspiration woven into this drink — whether it's to heal or to congregate — these tales point to a possible history and purpose of this drink. Today we pause and appreciate a rich tradition and meaningful history that will inspire us in the creation of the first connected device, dedicated to chai.
Infused not only with spices, milk, tea and sugar, but also mystique and history, each cup of chai can bring us the comfort of ancient times and deep meaning we could never imagine.
Have you heard of other mythical or familial stories about where chai came from? We'd love to hear your versions! Drop us a line on Twitter or Facebook.
Curious about our launch? Join our mailing list!


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