Validation of reference genes in a wide of samples Functional Plant Biology 343 enzyme 10 (UBC10)(Remans et al.2008).In a recent paper a germinated and grown under controlled conditions in pots in a Brassica napus L.microarray database was analysed,which climate room:25C day/20C night temperature,12h light/12h showed that EF/-a.and a new unknown protein I (UP1)were dark cycles.The plants were used to collect tissues under normal the most suitable reference genes among the given set of tissues growth conditions and after biotic and abiotic stress treatments. (Chen et al.2010).Furthermore,two commonly used reference All samples were snapped frozen in liquid nitrogen and kept genes ACTIN-7 and UBC21,plus two new genes,TIP41-like at-80°C until use. protein (TIP41)and PP2A that were selected from a microarray dataset,were identified as the most stable reference gene set for Developmental stages(Ds) normalisation during B.napus embryo maturation (Chen et al. Three young leaves per plant were harvested and leaves of three 2010).A study in Chinese cabbage showed that EF/-o and plants were pooled for each developmental stage:(i)early stage adenine phosphoribosyl-transferase (Apr)were the most stably (third leave present)(Ds1);(ii)before bolting (8 weeks after expressed genes among different tissues (root,stem,heading sowing)(Ds2);and (iii)after bolting (10 weeks after sowing) leaves and lateral sprout)(Qi et al.2010) Ds3). The morphological variation present within Brassica rapa (L.)Hanelt is enormous.This includes the leaves in crops like Different tissues (Dt) heading Chinese cabbages and the leafy types that do not form Eight different tissues including root(Dtl)and stem(Dt2)at the heads (pak choi,caixin and several Japanese vegetables like third leaf stage,leaves after bolting (Dt3,same sample as Ds3) mizuna,mibuna and komatsuna),the enlarged roots of turips, flower buds(Dt4),petiole s(Dt5),stamens(Dt6),pistils(Dt7)and the inflorescences and stems of broccoletto and the seeds of seed pods (Dt8),were collected from three plants and pooled. the oil types.When studying the genetic relationship among accessions using AFLP and SSR marker profiling,clusters or Biotic stress treatments (Bs) groups of accessions were identified that were represented by different crop types,but it was also clear that genetic Two fungi,Peronospora parasitica (P.p)and Alternaria distance was more defined by geographical origin than by crop brassicicola (A.b),were isolated from the leaves of different type (Zhao et al.2007,2010).There is no information about susceptible B.rapa cultivars in the farm of Nanjing selection of reference genes for nommalisation of gRT-PCR Agricultural University,China.Conidial suspensions were results for gene expression studies in Chinese cabbage.With adjusted to Ix 10%sporesmL and Tween-20 was added as a the recently released B.rapa genome sequence (The Brassica surfactant to a final concentration of 0.1%.For each treatment, rapa Genome Sequencing Project Consortium 2011)and 53-week-old seedlings were sprayed either with 50 mL pathogen development of gene expression platforms for B.rapa, suspension,or demi water (as control).Treated seedlings genome-wide large-scale gene expression studies will become were placed in a climate chamber (25C,85%+5%RH, available and will be mined to select reference genes for real 12 hour light/12 hour dark)and the second leaves from three time PCR studies. plants per treatment were harvested and pooled at 48h after In the present study,we selected and validated 13 reference inoculation.The two treatments are referred to as Bsl and Bs2 genes particularly for accurate normalisation ofgRT-PCR results respectively in non-heading Chinese cabbage.These reference genes include seven widely used reference genes in plant research (ACTIN, Abiotic stress treatments (As) ACTIN-2,GAPDH,Tub_a,CyP,EFI-a and 18S rRNA)and six Fifty seedlings (3 weeks old)were sprayed respectively with potential reference genes (ACTIN-1,UBO,UBC30,PPR,PP2A 50 mL solutions,water(as control),SA(2 mmolL,PH=6.5), and MDA)that were identified based on their stability in ABA(50 umol),NaCl(200 mM),H2O2(100 uM)and Mannitol expression studies comparing plant developmental stages, (400 mM).For salt (Asl)and drought (As2)stress treatments, different tissues or various environmental stimuli including leaves were harvested at 12 h(for NaCl and Mannitol)after stress biotic and abiotic stress (Brunner et al.2004;Czechowski treatments.For hormone treatments.leaves were harvested at et al.2005;Reid et al.2006).The 13 genes were tested in 20 6h after SA treatment(As3),24h after ABA treatment(As4) different samples,including three developmental stages,eight and 24 h after H2O2 treatment(As5).In addition,leaves from different tissues harvested at the mature plant developmental 50 seedlings(3 weeks old)that were exposed to cold(4C)and stage (between flowering and seed set)and from seedlings heat shock (40C)were harvested 2h after temperature stress exposed to two biotic stresses and seven abiotic stress treatments (As6 and As7). treatments,ranging from hormone-,salt-,drought-,till temperature stress treatments.We used the statistical algorithms RNA isolation,quality control and cDNA synthesis geNorm (Vandesompele et al.2002)and NormFinder (Andersen Total RNA was isolated by RNA simple Total RNA Kit et al.2004),which have been widely employed to select the extractions (Bio Teke.Beijing.China).Genomic DNA best suitable reference genes from given biological samples contaminations were effectively removed using RNase-free (Silver et al.2006;Wierschke et al.2010). DNase I treatment(Invitrogen,Carlsbad,CA,USA)according to manufacturer's instructions,as melting curve gave single peak and genomic amplification was larger than RNA/cDNA Materials and methods amplification(see Fig.S1,available as Supplementary Material Seeds of one pakchoi inbred line (Brassica rapa ssp.chinensis to this paper).RNA integrity was electrophoretically verified (L.)Hanelt;Suzhou Qing,a non-heading Chinese cabbage)were by agarose gel and by 260/280nm absorption ratio 1.9~2.1enzyme 10 (UBC10) (Remans et al. 2008). In a recent paper a Brassica napus L. microarray database was analysed, which showed that EF1-a and a new unknown protein 1 (UP1) were the most suitable reference genes among the given set of tissues (Chen et al. 2010). Furthermore, two commonly used reference genes ACTIN-7 and UBC21, plus two new genes, TIP41-like protein (TIP41) and PP2A that were selected from a microarray dataset, were identified as the most stable reference gene set for normalisation during B. napus embryo maturation (Chen et al. 2010). A study in Chinese cabbage showed that EF1-a and adenine phosphoribosyl-transferase (Apr) were the most stably expressed genes among different tissues (root, stem, heading leaves and lateral sprout) (Qi et al. 2010). The morphological variation present within Brassica rapa (L.) Hanelt is enormous. This includes the leaves in crops like heading Chinese cabbages and the leafy types that do not form heads (pak choi, caixin and several Japanese vegetables like mizuna, mibuna and komatsuna), the enlarged roots of turnips, the inflorescences and stems of broccoletto and the seeds of the oil types. When studying the genetic relationship among accessions using AFLP and SSR marker profiling, clusters or groups of accessions were identified that were represented by different crop types, but it was also clear that genetic distance was more defined by geographical origin than by crop type (Zhao et al. 2007, 2010). There is no information about selection of reference genes for normalisation of qRT–PCR results for gene expression studies in Chinese cabbage. With the recently released B. rapa genome sequence (The Brassica rapa Genome Sequencing Project Consortium 2011) and development of gene expression platforms for B. rapa, genome-wide large-scale gene expression studies will become available and will be mined to select reference genes for real time PCR studies. In the present study, we selected and validated 13 reference genes particularly for accurate normalisation of qRT–PCR results in non-heading Chinese cabbage. These reference genes include seven widely used reference genes in plant research (ACTIN, ACTIN-2, GAPDH, Tub_a, CyP, EF1-a and 18S rRNA) and six potential reference genes (ACTIN-1, UBQ, UBC30, PPR, PP2A and MDH) that were identified based on their stability in expression studies comparing plant developmental stages, different tissues or various environmental stimuli including biotic and abiotic stress (Brunner et al. 2004; Czechowski et al. 2005; Reid et al. 2006). The 13 genes were tested in 20 different samples, including three developmental stages, eight different tissues harvested at the mature plant developmental stage (between flowering and seed set) and from seedlings exposed to two biotic stresses and seven abiotic stress treatments, ranging from hormone-, salt-, drought-, till temperature stress treatments. We used the statistical algorithms geNorm (Vandesompele et al. 2002) and NormFinder (Andersen et al. 2004), which have been widely employed to select the best suitable reference genes from given biological samples (Silver et al. 2006; Wierschke et al. 2010). Materials and methods Seeds of one pakchoi inbred line (Brassica rapa ssp. chinensis (L.) Hanelt; Suzhou Qing, a non-heading Chinese cabbage) were germinated and grown under controlled conditions in pots in a climate room: 25
C day/20
C night temperature, 12 h light/12 h dark cycles. The plants were used to collect tissues under normal growth conditions and after biotic and abiotic stress treatments. All samples were snapped frozen in liquid nitrogen and kept at 80
C until use. Developmental stages (Ds) Three young leaves per plant were harvested and leaves of three plants were pooled for each developmental stage: (i) early stage (third leave present) (Ds1); (ii) before bolting (8 weeks after sowing) (Ds2); and (iii) after bolting (10 weeks after sowing) (Ds3). Different tissues (Dt) Eight different tissues including root (Dt1) and stem (Dt2) at the third leaf stage, leaves after bolting (Dt3, same sample as Ds3), flower buds (Dt4), petiole s(Dt5), stamens (Dt6), pistils (Dt7) and seed pods (Dt8), were collected from three plants and pooled. Biotic stress treatments (Bs) Two fungi, Peronospora parasitica (P.p) and Alternaria brassicicola (A.b), were isolated from the leaves of different susceptible B. rapa cultivars in the farm of Nanjing Agricultural University, China. Conidial suspensions were adjusted to 1  105 spores mL–1 and Tween-20 was added as a surfactant to a final concentration of 0.1%. For each treatment, 53-week-old seedlings were sprayed either with 50 mL pathogen suspension, or demi water (as control). Treated seedlings were placed in a climate chamber (25
C, 85%  5% RH, 12 hour light/12 hour dark) and the second leaves from three plants per treatment were harvested and pooled at 48 h after inoculation. The two treatments are referred to as Bs1 and Bs2 respectively. Abiotic stress treatments (As) Fifty seedlings (3 weeks old) were sprayed respectively with 50 mL solutions, water (as control), SA (2 mmol L1 , PH = 6.5), ABA (50 mmol), NaCl (200 mM), H2O2 (100 mM) and Mannitol (400 mM). For salt (As1) and drought (As2) stress treatments, leaves were harvested at 12 h (for NaCl and Mannitol) after stress treatments. For hormone treatments, leaves were harvested at 6 h after SA treatment (As3), 24 h after ABA treatment (As4) and 24 h after H2O2 treatment (As5). In addition, leaves from 50 seedlings (3 weeks old) that were exposed to cold (4
C) and heat shock (40
C) were harvested 2 h after temperature stress treatments (As6 and As7). RNA isolation, quality control and cDNA synthesis Total RNA was isolated by RNA simple Total RNA Kit extractions (Bio Teke, Beijing, China). Genomic DNA contaminations were effectively removed using RNase-free DNase I treatment (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions, as melting curve gave single peak and genomic amplification was larger than RNA/cDNA amplification (see Fig. S1, available as Supplementary Material to this paper). RNA integrity was electrophoretically verified by agarose gel and by 260/280 nm absorption ratio 1.9~2.1 Validation of reference genes in a wide of samples Functional Plant Biology 343