BACKGROUND
buckwheat; fagopyrum spp.
is a pseudo-cereal multipurpose food crop used for both grains and greens with several medicinal and nutritional properties  <cit> .
genus fagopyrum belongs to polygonaceae and has  <dig> known species which mainly occur in the highlands of euro-asia  <cit> .
out of these, two cultivated species, fagopyrum esculentum  and f. tataricum  are of high economic importance due to multiple uses such as a substitute for cereals in human consumption, as a vegetable crop, honey crop, and of ethno-botanical importance  <cit> .
significantly higher contents of flavonoids such as rutin and other polyphenols also add significance to the dietary value of buckwheat  <cit> .
in tartary buckwheat, fagopyritols; mono-, di- and trigalactosyl derivatives of d-chiro-inositol account for 40% of total soluble carbohydrates compared to 21% in common buckwheat thus, helps in the treatment of diabetes  <cit> .
total flavonoids are relatively higher in tartary buckwheat  compared to common buckwheat  of which rutin is the major component  <cit> .
rutin a major flavonoid of medicinal value is found in higher quantities in buckwheat thus, considered as a major dietary source of rutin  <cit> .
tartary buckwheat seeds contain more rutin  compared to common buckwheat seeds   <cit> .
due to the presence of proteins with high biological value  and flavonoids with higher concentration in tartary buckwheat compared to common buckwheat, the former is considered an excellent food material with a potential for preventive nutrition  <cit> .
but tartary buckwheat has a tightly adhering hull that makes it difficult to dehull and contains a bitter component that affects its palatability  <cit> .
however, rice-tartary is a type of tartary buckwheat  with a non-adhering hull property, and can be a potential nutraceutical food source  <cit> .
molecular basis of nutritional superiority, particularly higher rutin content in f. tataricum compared to f. esculentum, is not fully understood.
de-novo sequencing was used to understand molecular basis of morphological variations in the flowers of fagopyrum species  <cit> .
in addition, it has been observed that the flavonoid biosynthesis genes in f. esculentum were highly expressed in lower parts of plants than upper parts suggesting that flavonoids may be transported within a plant  <cit> .
anthocyanin content of f. tataricum has been correlated with the differential expression of flavonoid biosynthesis genes  <cit> .
comparative analysis of rutin content in different seed maturation stages of rice-tartary and tartary buckwheat compared to common buckwheat showed that all the post-flowering stages, s <dig>  s <dig>  s <dig> and s <dig> of rice-tartary contained  <dig> ,  <dig>   <dig>  and 43x higher rutin content compared to common buckwheat, respectively  <cit> .
stages s <dig>  s <dig>  and s <dig> of rice-tartary contained higher rutin content even compared to tartary buckwheat; figures  <dig> & <dig>  relatively higher expression of flavonoid pathway genes, phenylalanine ammonia lyase; pal  <dig> .
<dig> , chalcone synthase; chs  <dig> .
<dig> , chalcone isomerase; chi  <dig> .
<dig>  and flavonol synthase; fls  <dig> .
<dig>  were suggested to be responsible for higher rutin content in rice-tartary compared to common buckwheat  <cit> .
however, increase in the expression of pal, chs, chi and fls genes did not occur concomitant to an increase in rutin content.
therefore, identification of additional genes, if any, was carried out to investigate molecular basis of high rutin content in flowering and post-flowering stages of f. tataricum compared to f. esculentum.
flavonoid content in a particular tissue and developmental stage is largely influenced by different classes of regulatory genes, transporters, modifiers etc.
in addition to structural genes of the flavonoid biosynthetic pathway  <cit> .
lack of whole genome sequence and non-availability of ests from developing seeds of fagopyrum spp.
prompted us to utilize cdna-aflp to decipher what genetic factors contribute to nutritional superiority of rice-tartary buckwheat compared to common buckwheat.
present study reports several differentially expressed transcript fragments representing genes involved in basic and secondary metabolism, transcription factors, transporters, etc., which were validated through qrt-pcr to associate their contribution in nutritional superiority of rice-tartary over common buckwheat.
RESULTS
identification and analysis of differentially expressed transcripts
cdna-aflp analysis on rna samples from flower to mature seed stages of rice-tartary and common buckwheat with  <dig> primer pair combinations resulted in the identification of  <dig> clear and unambiguous fragments .
the tdfs ranged in sizes from 150–750 bp representing a total of  <dig>  tdfs.
a total of  <dig> differential tdfs based on presence/absence or differences in intensity were eluted from the gels, re-amplified and sequenced; figure  <dig>  dna sequence of each tdf was assigned a putative biological function by checking against the gene bank database  as well as the tair database; figure  <dig>  tdfs represented genes controlling different biological processes such as general and secondary metabolism , regulation , signal transduction , transportation , cellular organization , transposable elements , photosynthesis  and defense & response to stimuli ; additional file 1: table s <dig>  figure  <dig>
tdfs representing genes encoding alanine glyoxlate, methionine sulfoxide, fatty acid desaturase, kas iii, sucrose  <dig> phosphatase, ubiquitin protein ligases etc.
were implicated in the biosynthesis of proteins & amino acids , fatty acids  and carbohydrates .
similarly, 8% of tdfs corresponded to genes for key enzymes involved in secondary metabolism, including the flavonoid and anthocyanin biosynthesis.
tdfs  involved in cellular function included genes coding for pectin acyltransferases, proline rich extensins, glycine rich proteins, and arabinogalactan etc.
tdfs corresponding to transporters  included abc transporters, auxin hydrogen symporter, sugar transporters, zinc and potassium transporters.
genes involved in signal transduction  and regulation , including zn finger binding proteins, leucine rich repeats calmodulin binding protein, protein kinases and transcription factors belongs to myb and wrky classes were also detected.
tdfs representing differentially expressed genes were classified into different categories on the basis of their presence/absence  or differences in amount of expression  between both the fagopyrum species so as to infer whether tdfs belonging to a particular biological functional class are preferentially expressed in a particular fagopyrum species; figure 5a & b. tdfs representing genes involved in carbohydrate metabolism and signal transduction were relatively higher in number in f. esculentum, whereas tdfs representing genes involved in secondary metabolism, amino acid & protein metabolism, energy and photosynthesis were more in rice-tartary buckwheat; figure 5a.
most of the tdfs belonged to genes for transporters, transcriptional regulation, secondary metabolism, photosynthesis & energy, carbohydrate, protein and amino acid metabolism and showed relatively increased transcript abundance in rice-tartary compared to common buckwheat; figure 5b.
whereas, only tdfs for cellular metabolism showed relatively more transcript abundance in common buckwheat compared to rice-tartary buckwheat .
tdfs representing genes involved in transport of metabolites , regulation of biosynthesis , metabolism of metabolites , signal transduction  and energy transfer  were chosen for quantitative rt-pcr analysis in different seed maturing stages of both the fagopyrum species .
transcript quantification of selected tdfs in flower to seed maturation stages of fagopyrum spp
four tdfs representing genes for ubiquitin protein ligase , abc transporter , sugar transporter  and myb <dig>  showed significantly increased transcript abundance in flowers  of rice-tartary compared to common buckwheat with  <dig> ,  <dig> ,  <dig>  and  <dig>  folds expression, respectively; figure 6a.
relatively higher abundance was observed for tdfs from genes for abc transporter, sugar transporter, ub protein ligase and zn finger binding protein  in immature seed stage  of rice-tartary buckwheat with  <dig> ,  <dig> ,  <dig> ,  <dig>  folds, respectively over common buckwheat; figure 6b.
on the other hand,  <dig> tdfs from genes encoding zn finger binding protein, atp cf <dig> subunit , calmodulin binding protein, extensin  and auxin efflux  showed relatively increased transcript abundance in the immature seeds  of common buckwheat compared to rice-tartary buckwheat with  <dig> ,  <dig> ,  <dig> ,  <dig> , and  <dig>  folds higher abundance respectively.
two tdfs for abc transporter  and myb <dig>  showed relatively increased abundance in s <dig> of rice-tartary in comparison to common buckwheat; figure 6c.
in mature seeds , the expression of tdfs zn finger binding protein , abc transporter , ub protein ligase , calmodulin binding protein , and sugar transporter  was found to be higher in common compared to rice-tartary buckwheat.
whereas, extensin and atp cf <dig> subunit showed relatively higher expression in rice-tartary over common buckwheat in mature seeds  with  <dig>  and  <dig>  folds, respectively; figure 6d.
no transcript was detected for myb  <dig> and auxin efflux carrier protein in mature seeds of rice-tartary buckwheat.
therefore, the transcripts of ubiquitin protein ligases, abc transporter and sugar transporter showed relatively higher expression in three stages of seed development, including flowers and immature seeds of rice-tartary compared to common buckwheat.
discussion
understanding types and number of genes differentially expressed during seed maturation would help in discerning molecular mechanisms contributing to nutritional superiority, including high rutin content in the seeds of f. tataricum  over f. esculentum <cit> .
de-novo sequencing of transcripts from flowers of fagopyrum species  had shown that genes contributing to different biological processes are contributing to variations in the morphology of flowers in fagopyrum species  <cit> .
accumulation of higher amounts of rutin during post-flowering stages of f. tataricum  over f. esculentum has also been observed  <cit> .
overall nutritional superiority of f. tataricum over other fagopyrum spp has been found in the mature seeds as well as during seed maturation stages.
for example, increase in fagopyritols content was shown from immature to mature seeds of buckwheat  <cit> .
in addition, increase in the amount of sucrose and rutin was also reported during seed maturation of fagopyrum species  <cit> .
molecular dissection of the genetic machinery contributing to nutritional differences in the seeds of f. tataricum, in particular the rice-tartary type over f. esculentum  was undertaken through cdna-aflp since it is an open architecture technology for global transcriptional analysis in a non-model plant species  <cit> .
large numbers of tdfs with differential expression pattern were observed in different seed developmental stages of both the fagopyrum species.
tdfs with differential expression patterns belonged to genes involved in primary and secondary metabolism, transportation, signal transduction, gene regulation, photosynthesis & energy, defense and cellular processes in seed developmental stages of the fagopyrum spp.
; additional file 1: table s <dig>  out of  <dig> differential tdfs, only  <dig> showed 50–70% identity with the available floral transcriptome of both the fagopyrum species  <cit> , thereby, suggesting that most of the tdfs identified in the current study represented new genes.
tdfs involved in transport, transcription, secondary metabolism, amino acid & protein metabolism, carbohydrate metabolism and photosynthesis were relatively higher in number and expression pattern in rice-tartary over common buckwheat.
higher expression of tdfs involved in secondary metabolism and transportation such as chalcone synthase, dihydroflavonol reductase, udp glucosyl transferases, abc transporters, mate efflux carrier proteins, which are known to be involved in biosynthesis, accumulation and transportation of flavonoids, indicate their involvement in significantly higher flavonoid content in rice-tartary buckwheat  <cit> .
increased transcript abundance of tdf encoding lys/his transporter in rice-tartary buckwheat was implicated for higher amount of histidine in this species  <cit> .
tdfs involved in amino acid & protein metabolism , transcriptional regulation  and signal transduction  were also found to be abundant in rice-tartary buckwheat.
most of these tdfs represent genes with their direct or indirect role in controlling the growth and development of seeds and/or their nutritional composition.
on the basis of differential expression pattern of transcripts in rice-tartary and common buckwheat, the tdfs representing genes which have been implicated in biosynthesis, modification, regulation and transport of secondary metabolites  <cit>  were chosen to investigate their role, through qrt-pcr analysis, in the biosynthesis of higher rutin content in the seeds of rice-tartary over common buckwheat.
the flavonoid content increase in buckwheat seedlings has been attributed to the increase in the concentration of sucrose  <cit> .
in addition, sugars also act as developmental signals regulating seed maturation and accumulation of flavonoids in plants such as arabidopsis, v. vinifera <cit> .
exponential increase in the transcript of a sugar transporter  from flowers  to immature seeds  of rice-tartary compared to common buckwheat suggests its contribution in higher content of flavonoids and fagopyritols in the seeds of rice-tartary buckwheat.
relatively increased transcript abundance of auxin efflux carrier protein  in different seed maturation stages  of f. esculentum suggests its negative role in the biosynthesis of flavonoids, which are present in lower amounts in different growth stages of f. esculentum <cit> .
flavonoids have been implicated as inhibitors of auxin transport in arabidopsis <cit> .
the abc and mate classes of transporters are known to be involved in the transport of flavonoids from cytosol into vacuoles  <cit> .
in present study the abundance of tdf abc transporter  was relatively higher in flowering to seed maturation stages, s <dig> , s <dig> , and s <dig>  of rice-tartary buckwheat compared to common buckwheat suggested that this gene might be playing a key role in the transport of flavonoids  in rice-tartary buckwheat.
also, it has been shown that biosynthesis of flavonoids takes place in lower parts of fagopyrum spp.
and then gets transported to upper parts  <cit> .
significantly higher expression of  <dig> tdfs representing genes for ubiquitin protein ligase, abc transporter, sugar transporter and calmodulin binding protein in s <dig> of rice-tartary buckwheat compared to f. esculentum suggests their major involvement in nutritional superiority of rice-tartary buckwheat.
the calmodulin binding proteins regulate diverse cellular processes by interacting with other proteins and help in secondary metabolism by acting as secondary messengers through signal transduction  <cit> .
in addition, calmodulin proteins are also known to induce anthocyanin biosynthesis in v. vinifera <cit> .
extensins, the major structural proteins in plant cell wall play important role in various biological processes such as embryo development, root hair growth, seed coat development, defense, etc.
<cit> .
increased transcript abundance of extensin protein  in mature seeds of rice-tartary buckwheat in comparison to common buckwheat may contribute to development of seeds of both the fagopyrum species.
transcription factors are known to play important role in various seed development processes and regulation of secondary metabolism  <cit> .
zn finger binding proteins  have been implicated in regulation of important biological processes such as flower and seed development, seed germination, stress tolerance in arabidopsis <cit> .
expression of a tdf representing a gene for zn finger binding protein was relatively higher in seed developing stages s <dig> and s <dig> of common buckwheat compared to rice-tartary buckwheat.
transcript of a tdf encoding for another transcription factor myb  <dig>  was high in the flowers  of rice-tartary compared to common buckwheat.
ub protein ligases  are known to regulate various biological processes like photomorphogenesis, hormonal response, senescence, floral, embryo and seedling development through degradation of proteins as well as regulates phenylpropanoid pathway during uv stress and sugar signaling during seedling development  <cit> .
relatively higher expression of ub protein ligase in immature seeds  of rice-tartary buckwheat has been observed in the present study.
significantly higher expression of most of the selected tdfs during early seed formation stage  of rice-tartary buckwheat  reflects their biological importance in maintaining higher amounts of rutin, which otherwise drops significantly in the same stage of f. esculentum.
CONCLUSIONS
the present study concludes that in addition to structural genes, the other classes of genes such as regulators, modifiers and transporters are equally important in contributing to higher flavonoids content and nutritional superiority of f. tataricum  over f. esculentum.
increased transcript abundance of selected tdfs in rice-tartary buckwheat during early seed maturation stage  i.e.
the transition from flowers to seed formation also reflects their contribution not only in higher rutin content but also in other biological processes which are contributing to overall nutritional differences between both the fagopyrum species.
in summary, the cdna-aflp technology was successfully utilized to identify genes that are differentially expressed in seed maturation stages of two fagopyrum spp.
therefore, identification of several genes representing regulators, modifiers or transporters, has opened up avenues to investigate their precise role in contributing to higher rutin content as well as overall nutritional superiority of rice-tartary over common buckwheat.

 buckwheat; fagopyrum spp. is a pseudo-cereal multipurpose food crop used for both grains and greens with several medicinal and nutritional properties. genus fagopyrum belongs to polygonaceae and has known species which mainly occur in the highlands of euro-asia. out of these, two cultivated species, fagopyrum esculentum and f. tataricum are of high economic importance due to multiple uses such as a substitute for cereals in human consumption, as a vegetable crop, honey crop, and of ethno-botanical importance. significantly higher contents of flavonoids such as rutin and other polyphenols also add significance to the dietary value of buckwheat. in tartary buckwheat, fagopyritols; mono-, di- and trigalactosyl derivatives of d-chiro-inositol account for 40% of total soluble carbohydrates compared to 21% in common buckwheat thus, helps in the treatment of diabetes. total flavonoids are relatively higher in tartary buckwheat compared to common buckwheat of which rutin is the major component. rutin a major flavonoid of medicinal value is found in higher quantities in buckwheat thus, considered as a major dietary source of rutin. tartary buckwheat seeds contain more rutin compared to common buckwheat seeds. due to the presence of proteins with high biological value and flavonoids with higher concentration in tartary buckwheat compared to common buckwheat, the former is considered an excellent food material with a potential for preventive nutrition. but tartary buckwheat has a tightly adhering hull that makes it difficult to dehull and contains a bitter component that affects its palatability. however, rice-tartary is a type of tartary buckwheat with a non-adhering hull property, and can be a potential nutraceutical food source. molecular basis of nutritional superiority, particularly higher rutin content in f. tataricum compared to f. esculentum, is not fully understood. de-novo sequencing was used to understand molecular basis of morphological variations in the flowers of fagopyrum species. in addition, it has been observed that the flavonoid biosynthesis genes in f. esculentum were highly expressed in lower parts of plants than upper parts suggesting that flavonoids may be transported within a plant. anthocyanin content of f. tataricum has been correlated with the differential expression of flavonoid biosynthesis genes. comparative analysis of rutin content in different seed maturation stages of rice-tartary and tartary buckwheat compared to common buckwheat showed that all the post-flowering stages, s s s and s of rice-tartary contained, and 43x higher rutin content compared to common buckwheat, respectively. stages s s and s of rice-tartary contained higher rutin content even compared to tartary buckwheat; figures  & relatively higher expression of flavonoid pathway genes, phenylalanine ammonia lyase; pal., chalcone synthase; chs., chalcone isomerase; chi. and flavonol synthase; fls. were suggested to be responsible for higher rutin content in rice-tartary compared to common buckwheat. however, increase in the expression of pal, chs, chi and fls genes did not occur concomitant to an increase in rutin content. therefore, identification of additional genes, if any, was carried out to investigate molecular basis of high rutin content in flowering and post-flowering stages of f. tataricum compared to f. esculentum. flavonoid content in a particular tissue and developmental stage is largely influenced by different classes of regulatory genes, transporters, modifiers etc. in addition to structural genes of the flavonoid biosynthetic pathway. lack of whole genome sequence and non-availability of ests from developing seeds of fagopyrum spp. prompted us to utilize cdna-aflp to decipher what genetic factors contribute to nutritional superiority of rice-tartary buckwheat compared to common buckwheat. present study reports several differentially expressed transcript fragments representing genes involved in basic and secondary metabolism, transcription factors, transporters, etc., which were validated through qrt-pcr to associate their contribution in nutritional superiority of rice-tartary over common buckwheat.  identification and analysis of differentially expressed transcripts cdna-aflp analysis on rna samples from flower to mature seed stages of rice-tartary and common buckwheat with primer pair combinations resulted in the identification of clear and unambiguous fragments. the tdfs ranged in sizes from 150–750 bp representing a total of tdfs. a total of differential tdfs based on presence/absence or differences in intensity were eluted from the gels, re-amplified and sequenced; figure dna sequence of each tdf was assigned a putative biological function by checking against the gene bank database as well as the tair database; figure tdfs represented genes controlling different biological processes such as general and secondary metabolism, regulation, signal transduction, transportation, cellular organization, transposable elements, photosynthesis and defense & response to stimuli; additional file 1: table s figure tdfs representing genes encoding alanine glyoxlate, methionine sulfoxide, fatty acid desaturase, kas iii, sucrose phosphatase, ubiquitin protein ligases etc. were implicated in the biosynthesis of proteins & amino acids, fatty acids and carbohydrates. similarly, 8% of tdfs corresponded to genes for key enzymes involved in secondary metabolism, including the flavonoid and anthocyanin biosynthesis. tdfs involved in cellular function included genes coding for pectin acyltransferases, proline rich extensins, glycine rich proteins, and arabinogalactan etc. tdfs corresponding to transporters included abc transporters, auxin hydrogen symporter, sugar transporters, zinc and potassium transporters. genes involved in signal transduction and regulation, including zn finger binding proteins, leucine rich repeats calmodulin binding protein, protein kinases and transcription factors belongs to myb and wrky classes were also detected. tdfs representing differentially expressed genes were classified into different categories on the basis of their presence/absence or differences in amount of expression between both the fagopyrum species so as to infer whether tdfs belonging to a particular biological functional class are preferentially expressed in a particular fagopyrum species; figure 5a & b. tdfs representing genes involved in carbohydrate metabolism and signal transduction were relatively higher in number in f. esculentum, whereas tdfs representing genes involved in secondary metabolism, amino acid & protein metabolism, energy and photosynthesis were more in rice-tartary buckwheat; figure 5a. most of the tdfs belonged to genes for transporters, transcriptional regulation, secondary metabolism, photosynthesis & energy, carbohydrate, protein and amino acid metabolism and showed relatively increased transcript abundance in rice-tartary compared to common buckwheat; figure 5b. whereas, only tdfs for cellular metabolism showed relatively more transcript abundance in common buckwheat compared to rice-tartary buckwheat. tdfs representing genes involved in transport of metabolites, regulation of biosynthesis, metabolism of metabolites, signal transduction and energy transfer were chosen for quantitative rt-pcr analysis in different seed maturing stages of both the fagopyrum species. transcript quantification of selected tdfs in flower to seed maturation stages of fagopyrum spp four tdfs representing genes for ubiquitin protein ligase, abc transporter, sugar transporter and myb showed significantly increased transcript abundance in flowers of rice-tartary compared to common buckwheat with,, and folds expression, respectively; figure 6a. relatively higher abundance was observed for tdfs from genes for abc transporter, sugar transporter, ub protein ligase and zn finger binding protein in immature seed stage of rice-tartary buckwheat with,,, folds, respectively over common buckwheat; figure 6b. on the other hand, tdfs from genes encoding zn finger binding protein, atp cf subunit, calmodulin binding protein, extensin and auxin efflux showed relatively increased transcript abundance in the immature seeds of common buckwheat compared to rice-tartary buckwheat with,,,, and folds higher abundance respectively. two tdfs for abc transporter and myb showed relatively increased abundance in s of rice-tartary in comparison to common buckwheat; figure 6c. in mature seeds, the expression of tdfs zn finger binding protein, abc transporter, ub protein ligase, calmodulin binding protein, and sugar transporter was found to be higher in common compared to rice-tartary buckwheat. whereas, extensin and atp cf subunit showed relatively higher expression in rice-tartary over common buckwheat in mature seeds with and folds, respectively; figure 6d. no transcript was detected for myb and auxin efflux carrier protein in mature seeds of rice-tartary buckwheat. therefore, the transcripts of ubiquitin protein ligases, abc transporter and sugar transporter showed relatively higher expression in three stages of seed development, including flowers and immature seeds of rice-tartary compared to common buckwheat. discussion understanding types and number of genes differentially expressed during seed maturation would help in discerning molecular mechanisms contributing to nutritional superiority, including high rutin content in the seeds of f. tataricum over f. esculentum. de-novo sequencing of transcripts from flowers of fagopyrum species had shown that genes contributing to different biological processes are contributing to variations in the morphology of flowers in fagopyrum species. accumulation of higher amounts of rutin during post-flowering stages of f. tataricum over f. esculentum has also been observed. overall nutritional superiority of f. tataricum over other fagopyrum spp has been found in the mature seeds as well as during seed maturation stages. for example, increase in fagopyritols content was shown from immature to mature seeds of buckwheat. in addition, increase in the amount of sucrose and rutin was also reported during seed maturation of fagopyrum species. molecular dissection of the genetic machinery contributing to nutritional differences in the seeds of f. tataricum, in particular the rice-tartary type over f. esculentum was undertaken through cdna-aflp since it is an open architecture technology for global transcriptional analysis in a non-model plant species. large numbers of tdfs with differential expression pattern were observed in different seed developmental stages of both the fagopyrum species. tdfs with differential expression patterns belonged to genes involved in primary and secondary metabolism, transportation, signal transduction, gene regulation, photosynthesis & energy, defense and cellular processes in seed developmental stages of the fagopyrum spp.; additional file 1: table s out of differential tdfs, only showed 50–70% identity with the available floral transcriptome of both the fagopyrum species, thereby, suggesting that most of the tdfs identified in the current study represented new genes. tdfs involved in transport, transcription, secondary metabolism, amino acid & protein metabolism, carbohydrate metabolism and photosynthesis were relatively higher in number and expression pattern in rice-tartary over common buckwheat. higher expression of tdfs involved in secondary metabolism and transportation such as chalcone synthase, dihydroflavonol reductase, udp glucosyl transferases, abc transporters, mate efflux carrier proteins, which are known to be involved in biosynthesis, accumulation and transportation of flavonoids, indicate their involvement in significantly higher flavonoid content in rice-tartary buckwheat. increased transcript abundance of tdf encoding lys/his transporter in rice-tartary buckwheat was implicated for higher amount of histidine in this species. tdfs involved in amino acid & protein metabolism, transcriptional regulation and signal transduction were also found to be abundant in rice-tartary buckwheat. most of these tdfs represent genes with their direct or indirect role in controlling the growth and development of seeds and/or their nutritional composition. on the basis of differential expression pattern of transcripts in rice-tartary and common buckwheat, the tdfs representing genes which have been implicated in biosynthesis, modification, regulation and transport of secondary metabolites were chosen to investigate their role, through qrt-pcr analysis, in the biosynthesis of higher rutin content in the seeds of rice-tartary over common buckwheat. the flavonoid content increase in buckwheat seedlings has been attributed to the increase in the concentration of sucrose. in addition, sugars also act as developmental signals regulating seed maturation and accumulation of flavonoids in plants such as arabidopsis, v. vinifera. exponential increase in the transcript of a sugar transporter from flowers to immature seeds of rice-tartary compared to common buckwheat suggests its contribution in higher content of flavonoids and fagopyritols in the seeds of rice-tartary buckwheat. relatively increased transcript abundance of auxin efflux carrier protein in different seed maturation stages of f. esculentum suggests its negative role in the biosynthesis of flavonoids, which are present in lower amounts in different growth stages of f. esculentum. flavonoids have been implicated as inhibitors of auxin transport in arabidopsis. the abc and mate classes of transporters are known to be involved in the transport of flavonoids from cytosol into vacuoles. in present study the abundance of tdf abc transporter was relatively higher in flowering to seed maturation stages, s, s, and s of rice-tartary buckwheat compared to common buckwheat suggested that this gene might be playing a key role in the transport of flavonoids in rice-tartary buckwheat. also, it has been shown that biosynthesis of flavonoids takes place in lower parts of fagopyrum spp. and then gets transported to upper parts. significantly higher expression of tdfs representing genes for ubiquitin protein ligase, abc transporter, sugar transporter and calmodulin binding protein in s of rice-tartary buckwheat compared to f. esculentum suggests their major involvement in nutritional superiority of rice-tartary buckwheat. the calmodulin binding proteins regulate diverse cellular processes by interacting with other proteins and help in secondary metabolism by acting as secondary messengers through signal transduction. in addition, calmodulin proteins are also known to induce anthocyanin biosynthesis in v. vinifera. extensins, the major structural proteins in plant cell wall play important role in various biological processes such as embryo development, root hair growth, seed coat development, defense, etc.. increased transcript abundance of extensin protein in mature seeds of rice-tartary buckwheat in comparison to common buckwheat may contribute to development of seeds of both the fagopyrum species. transcription factors are known to play important role in various seed development processes and regulation of secondary metabolism. zn finger binding proteins have been implicated in regulation of important biological processes such as flower and seed development, seed germination, stress tolerance in arabidopsis. expression of a tdf representing a gene for zn finger binding protein was relatively higher in seed developing stages s and s of common buckwheat compared to rice-tartary buckwheat. transcript of a tdf encoding for another transcription factor myb was high in the flowers of rice-tartary compared to common buckwheat. ub protein ligases are known to regulate various biological processes like photomorphogenesis, hormonal response, senescence, floral, embryo and seedling development through degradation of proteins as well as regulates phenylpropanoid pathway during uv stress and sugar signaling during seedling development. relatively higher expression of ub protein ligase in immature seeds of rice-tartary buckwheat has been observed in the present study. significantly higher expression of most of the selected tdfs during early seed formation stage of rice-tartary buckwheat reflects their biological importance in maintaining higher amounts of rutin, which otherwise drops significantly in the same stage of f. esculentum.  the present study concludes that in addition to structural genes, the other classes of genes such as regulators, modifiers and transporters are equally important in contributing to higher flavonoids content and nutritional superiority of f. tataricum over f. esculentum. increased transcript abundance of selected tdfs in rice-tartary buckwheat during early seed maturation stage i.e. the transition from flowers to seed formation also reflects their contribution not only in higher rutin content but also in other biological processes which are contributing to overall nutritional differences between both the fagopyrum species. in summary, the cdna-aflp technology was successfully utilized to identify genes that are differentially expressed in seed maturation stages of two fagopyrum spp. therefore, identification of several genes representing regulators, modifiers or transporters, has opened up avenues to investigate their precise role in contributing to higher rutin content as well as overall nutritional superiority of rice-tartary over common buckwheat. 
the tdfs represented genes controlling different biological processes such as basic and secondary metabolism, regulation, signal transduction, transportation, cellular organization, and photosynthesis & energy. vegetative tissues of both the fagopyrum species contain almost similar amount of rutin; however, rutin content in seed of f. tataricum are ~ folds of that in seed of f. esculentum. in order to understand the molecular basis of high rutin content in f. tataricum, differential transcript profiling through cdna-aflp has been utilized to decipher what genetic factors in addition to flavonoid structural genes contribute to high rutin content of f. tataricum compared to f. esculentum. tdfs were then eluted, cloned and sequenced from f. tataricum and f. esculentum. most of the tdfs except belonging to cellular metabolism showed relatively higher transcript abundance in f. tataricum over f. esculentum. quantitative rt-pcr analysis of nine tdfs representing genes involved in regulation, metabolism, signaling and transport of secondary metabolites showed that all the tested nine tdfs except myb showed significantly higher expression in early seed formation stage of f. tataricum compared to f. esculentum. increased transcript abundance of tdfs during transition from flowers to seed maturation suggests their involvement not only in the higher rutin content of f. tataricum over f. esculentum but also in nutritional superiority of the former. qrt-pcr results were found to be consistent with the cdna-aflp results. the present study concludes that in addition to structural genes, other classes of genes such as regulators, modifiers and transporters are also important in biosynthesis and accumulation of flavonoid content in plants. buckwheat, consisting of two cultivated species fagopyrum tataricum and f. esculentum, is the richest source of flavonoid rutin. categorization of tdfs on the basis of their presence/absence or differences in the amount of expression between both the fagopyrum species showed that majority of variants are quantitative. cdna-aflp technology was successfully utilized to capture genes that are contributing to differences in rutin content in seed maturing stages of fagopyrum species.
 buckwheat, consisting of two cultivated species fagopyrum tataricum and f. esculentum, is the richest source of flavonoid rutin. vegetative tissues of both the fagopyrum species contain almost similar amount of rutin; however, rutin content in seed of f. tataricum are ~ folds of that in seed of f. esculentum. in order to understand the molecular basis of high rutin content in f. tataricum, differential transcript profiling through cdna-aflp has been utilized to decipher what genetic factors in addition to flavonoid structural genes contribute to high rutin content of f. tataricum compared to f. esculentum.  differential transcript profiling through cdna-aflp in seed maturing stages with primer combinations generated total of transcript fragments. tdfs were then eluted, cloned and sequenced from f. tataricum and f. esculentum. categorization of tdfs on the basis of their presence/absence or differences in the amount of expression between both the fagopyrum species showed that majority of variants are quantitative. the tdfs represented genes controlling different biological processes such as basic and secondary metabolism, regulation, signal transduction, transportation, cellular organization, and photosynthesis & energy. most of the tdfs except belonging to cellular metabolism showed relatively higher transcript abundance in f. tataricum over f. esculentum. quantitative rt-pcr analysis of nine tdfs representing genes involved in regulation, metabolism, signaling and transport of secondary metabolites showed that all the tested nine tdfs except myb showed significantly higher expression in early seed formation stage of f. tataricum compared to f. esculentum. qrt-pcr results were found to be consistent with the cdna-aflp results.  the present study concludes that in addition to structural genes, other classes of genes such as regulators, modifiers and transporters are also important in biosynthesis and accumulation of flavonoid content in plants. cdna-aflp technology was successfully utilized to capture genes that are contributing to differences in rutin content in seed maturing stages of fagopyrum species. increased transcript abundance of tdfs during transition from flowers to seed maturation suggests their involvement not only in the higher rutin content of f. tataricum over f. esculentum but also in nutritional superiority of the former. rutincdna-aflptranscript derived fragmentsqrt-pcr 