Ana səhifə

Braving the attitude of altitude: Caragana jubata at work in cold desert of Himalaya. Authors


Yüklə 1.52 Mb.
səhifə1/8
tarix10.05.2016
ölçüsü1.52 Mb.
  1   2   3   4   5   6   7   8
Braving the attitude of altitude: Caragana jubata at work in cold desert of Himalaya.

Authors:

Pardeep Kumar Bhardwaj1,2, Ritu Kapoor1, Deep Mala1, Geetika Bhagwat1, Vishal Acharya1, Anil Kumar Singh1, Surender Kumar Vats3, Paramvir Singh Ahuja1, Sanjay Kumar1*



1Biotechnology Division, 3Biodiversity Division, CSIR-Institute of Himalayan Bioresource Technology,

Council of Scientific and Industrial Research, P.O. Box 6, Palampur (H.P.) 176061, India



2Current address: Regional Centre of Institute of Bioresources and Sustainable Development (DBT), Tadong, Gangtok-737102, Sikkim, India

*Correspondence and requests for materials should be addressed to S.K.:



sanjaykumar@ihbt.res.in; sanjayplp@rediffmail.com.

Supplementary Figure S1. Graphical representation of expressed sequence tag (EST) distribution in low temperature (4°C, panel ‘a’) and control (25°C, panel ‘b’) subtracted libraries. Number of ESTs present in each category is shown in the parenthesis. Percent value shows relative occurrence out of the total ESTs. All the ESTs were functionally classified according to Munich Information Center for Protein Sequences (MIPS, http://www.helmholtz-muenchen.de/en/ibis).



Supplementary Figure S2. Expression analysis of 22 genes in response to low temperature (LT, 4°C) and the altitude by reverse transcriptase polymerase chain reaction (RT-PCR). All panels under ‘a’ exhibit gene expression at CO (control, 25°C) and LT in Caragana jubata. Panels under ‘b’ denote the gene expression in two populations (P1, and P2) of C. jubata growing in niche environment of high altitude (HA) in Kibber-Gete area (Lahaul and Spiti district, Himachal Pradesh, India) during September/October for two separate years. RT-PCR was also performed for the plants growing at low altitude (LA, 25°C) in the institute (Palampur, Himachal Pradesh, India). For RT-PCR, total RNA was treated with DNase I (amplification grade, Invitrogen, USA) to remove contaminating genomic DNA. First-strand cDNA was synthesized using 2μg of DNA-free RNA, Superscript III (Invitrogen, USA) and oligo (dT)12-18 (Invitrogen, USA). PCR was performed with 1 μl of first-strand cDNA and 0.5 μM each of forward and reverse gene specific primers in a final volume of 25 μl. Various primers and PCR conditions are listed in Supplementary Table S7. PCR products were separated on 1.2% agarose gel, stained with ethidium bromide and visualized using a gel documentation system (Alpha DigiDocTM, Alpha Innotech, USA). Amplicons were quantified using Alpha DigiDoc 1000 software by measuring integrated density value (IDV). Expression of 26S rRNA served as internal control1.



Supplementary Figure S3. Expression of 22 genes by quantitative real-time PCR (qRT-PCR) analysis in C. jubata growing at low and high altitudes. The first strand cDNA template was synthesized as described in Supplementary Figure S2, diluted 1:10 with nuclease-free water before use. Amplification was carried out with SYBR® Premix Ex TaqTM (Perfect Real-Time) (TaKaRa, China) and primers (Supplementary Table S7) on a Mx3000P Real-Time PCR System (Agilent Technologies, USA). Manufacturer’s instructions were followed wherever necessary. Three technical replicates were performed for each experiment. The 26S rRNA served as internal control and relative transcript abundance was calculated using REST 2009 software2.

Supplementary Figure S4. Expression analysis of gene homologues in Arabidopsis in response to low temperature (LT; 4°C). Gene homologue in Arabidopsis was identified through BLASTX in TAIR 10 (www.arabidopsis.org; Supplementary Table 4). The microarray data for genes of Arabidopsis under cold stress was retrieved from AtGenExpress (http://jsp.weigelworld.org/expviz/expviz.jsp). Datasets obtained were corresponding to LT stress at different time points viz. 0.5, 1, 3, 6, 12, and 24 h for aerial tissues and analysed with respect to control. Name of the Caragana jubata homologue gene is shown below the panel followed by corresponding Arabidopsis gene accession number.



Supplementary Figure S5. Expression analysis of gene homologues in rice in response to low temperature (LT; 4°C). Gene homologues in rice were identified through BLASTX in RGAP 73 (Supplementary Table 5). The microarray data of rice genes under cold stress (experiment ID GSE6901) was retrieved from rice oligonucleotide array database (www.ricearray.org). Datasets obtained were corresponding to LT (4°C) for 3 h and analysed with respect to control (28°C). The values were log2 transformed and used to plot graphs for gene expression. Name of the Caragana jubata homologue gene is shown below the panel; this was followed by corresponding rice gene accession number.



Supplementary Figure S6. Schematic diagram of acclimation experiment for Caragana jubata. Twelve weeks old plants were grouped into three sets: control (CO; plants exposed to 25°C throughout the experiment), non-acclimated [NA, plants initially exposed to 25°C for 120 h followed by exposure to LT (4°C) upto 192 h; thereafter, these plants were exposed to 25°C again for 48 h], and acclimated (ACC; plants exposed to a cycle of 4°C, 25°C, 4°C, 25°C, at 0 h, 72 h, 120 h and 192 h, respectively).



Supplementary Figure S7. Habitat of Caragana jubata in the high altitude cold desert niche at Kibber-Gete (Lahaul and Spiti district, Himachal Pradesh, India, panel ‘a’) along with a close up view of the plant (panel ‘b’).



Supplementary Figure S8. Differential screenings of the clones obtained from forward suppression subtractive hybridization library (SSHL). Forward SSHL (CjSF) represents the putative over-expressed ESTs in response to LT (4°C for 48h). ESTs from CjSF were blotted in duplicate onto nylon membranes. These membranes were hybridized with radiolabelled forward probe derived from the forward unsubtracted cDNA (4°C for 48 h; tester cDNA), and the reverse probe that was derived from reverse unsubtracted cDNA (25°C; driver cDNA). The numeral in between the blots corresponds to the clone ID as shown in Supplementary Table 2.



Supplementary Figure S9. Differential screenings of the clones obtained from reverse suppression subtractive hybridization library (SSHL). Reverse SSHL (CjSR) represents the over-expressed ESTs at CO (25°C). ESTs from CjSR were blotted in duplicate onto nylon membranes. These membranes were hybridized with radiolabelled forward probe derived from the forward unsubtracted cDNA (4°C for 48 h; tester cDNA), and reverse probe derived from reverse unsubtracted cDNA (25°C; driver cDNA). The numeral in between the blots corresponds to the clone ID as shown in Supplementary Table S3. Clone ID numerals 2, 11, 20, 44, 57, 98, 106, 112, 187, 220, 223, 234, 378, 365, 397, 424, 428, 436 are the genes related to photosynthesis that exhibit down-regulation at LT.

Supplementary Table S1. Summary of the forward (CjSF) and reverse (CjSR) suppression subtractive hybridization cDNA libraries. CjSF represents the putative expressed sequence tags (ESTs) possibly over-expressed in response to low temperature (4°C), whereas CjSR represents the putative ESTs over-expressed in control (25°C). Selected up- and down-regulated genes were finally validated by RT-PCR analysis.


Descriptive category

CjSF

CjSR

No. of Clones isolated/sequenced

556

560

No. of high quality ESTs

504

517

No. of ESTs selected

502

515

No. of Singletons

256

297

No. of Contigs

36

34

No. of putative Transcripts (UniESTs)

No. of ESTs in contigs

292

246


331

218


Average insert size (bp)

514

564

Contig EST redundancy (%)a

49.00

42.33
  1   2   3   4   5   6   7   8


Verilənlər bazası müəlliflik hüququ ilə müdafiə olunur ©anasahife.org 2016
rəhbərliyinə müraciət