Supplementary Information - Research Square

Supplementary Information

Research Article

Genome-wide identification and characterization of functionally relevant microsatellites from putative transcription factor genes of tea

for efficient genotyping.

Rajni Parmara,b, Romit Setha, Ram Kumar Sharmaa,b*

aBiotechnology Department, CSIR-Institute of Himalayan Bioresource Technology (CSIR-IHBT), Palampur, Himachal Pradesh, 176061, India

bAcademy of Scientific and Innovative Research (AcSIR), CSIR-IHBT, Palampur, Himachal Pradesh, 176061, India

*Correspondence:

Dr Ram Kumar Sharma

Biotechnology Division

CSIR-Institute of Himalayan Bioresource Technology

Palampur (H.P)

India

Email: [email protected]

mailto:[email protected]

Table S1: List of random genotypes utilized for screening of TTFDMS and TTFMS markers.

S.No. Genotypes Varietal Type

Source Remarks

1 TV 1 Assam China hybrid

Tocklai Experiment Station, Jorhat,Assam

Average yield and Quality, Moderately Susceptible to Drought

2 TV 19 Cambod Tocklai Experiment Station, Jorhat,Assam

High Yield and Quality, Drought Tolerant

3 SA 6 Assam UPASI Blister Blight Tolerant, Avg quality and yield

4 ASHA China Hybrid CSK HP KV Palampur

Blister Blight Sensitive, Quality

5 TS 449 Assam Hybrid

Tocklai Experiment Station, Jorhat,Assam

High Quality But Average Yield, Drought Tolerant

6 C 6017 Cambod Craigmore, The Nilgiris

Susceptible to Drought

7 TEENALI Assam Teenali Assam

8 U 9 Assam Brookland High Yielding, Moderately resistant to blister blight

Table S2: List of 115 genotypes (China hybrid) utilized for functional genetic diversity characterization using TTFMS markers.

S.No. Genotype Varietal Type Soruce

1 Baijnath1 China Hybrid Baijnath Tea corp.

2 BGP146 China Hybrid Banuri Tea germplasm,IHBT


4 BS12 China Hybrid Banuri Tea germplasm,IHBT


















22 CEF01 China Hybrid Dharamshala collection


24 CPF01 China Hybrid Dharamshala collection

25 PATTA01 China Hybrid Patta Tea Estate collection

26 SBARI02 China Hybrid Sidhbari Tea Estate collection

27 Saloh02 China Hybrid Saloh Tea Estate collection

28 SB05 China Hybrid Sidhbari Tea Estate collection

29 B668 China Hybrid Darjeeling clone, TRA



32 BGP 121 China Hybrid Banuri Tea germplasm,IHBT























55 BS 11 China Hybrid Banuri Tea germplasm,IHBT








63 BS 44 China Hybrid Banuri Tea germplasm,IHBT















78 Hoodle01 China hybrid Dharamshala collection

79 KangraAsha China Hybrid HPKV, Palampur

80 KangraJat China Hybrid Kangra region collection

81 Khalet05 China Hybrid Khalet region collection

82 Lahla01 China Hybrid Wah tea Estate collection

83 Mbal10 China Hybrid Mansimbal collection


85 Mpat02 China Hybrid Baijnath Tea Corp





90 Rajpur04 China Hybrid Rajpura Tea Estate collection

91 Saloh01 China Hybrid Saloh Tea Estate collection

92 T383 China Hybrid Darjeeling clone, TRA collection

93 BHATTU22 China Hybrid -





















114 Sbari04 China Hybrid Dharamshala collection

115 TH09 China Hybrid Thurbo Tea Estate Darjeeling

Figure S1: Overall repeat frequency of SSRs (Sequence complementary) in transcripts encoding

repeats (c) Tetra-repeats (e) Penta-repeats (f) Pie chart showing abundance of repeats

Overall repeat frequency of SSRs (Sequence complementary) in transcripts encoding putative Transcription Factor (TFs)

repeats (f) Pie chart showing abundance of repeats

Transcription Factor (TFs) (a) Di-repeats (b) Tri-

Figure S2: Distribution of SSR motifs in the ORF Region of the putative TFs gene in Tea

of SSR motifs in the ORF Region of the putative TFs gene in Tea (Di, Tri, Tetra, Penta and Hexa repeats motifs).

(Di, Tri, Tetra, Penta and Hexa repeats motifs).

Table S3: List of 15 Polymorphic SSR Markers from putative of Transcription Factor gene used for tea genotyping

S.No. Primer Name Ann Temp. Obs. Product Size Alleles Localisation TF Family

1 TTFDMS_39438 55 230-250 2 CDS Trihelix

2 TTFDMS_67364 55 170:180 2 CDS bHLH

3 TTFDMS_2467 55 200:250 2 CDS bHLH

4 TTFDMS_790 55 700:800 2 CDS MYB

5 TTFMS_7650 58 400:500 2 3' UTR bHLH

6 TTFMS_9298_A 55 250:300 2 3' UTR C3H

7 TTFMS_17457 55 200:250 2 3' UTR CPP

8 TTFMS_73176 55 150:200 2 3' UTR ERF

9 TTFMS_17070 58 250:300 2 3' UTR SBP

10 TTFMS_653 55 200:300 2 3' UTR STAT

11 TTFMS_30069 58 400:500 2 5' UTR C3H

12 TTFMS_1609 55 100:200 2 5' UTR FAR1

13 TTFMS_23075 55 350:400 2 5' UTR WRKY

14 TTFMS_7132 55 250:350 2 5' UTR C3H

15 TTFMS_30134 55 600:700 2 5' UTR FAR1

Figure S3a: GO categorisation of the putative TF genes of TeaGO categorisation of the putative TF genes of Tea (a) Biological process, (b) Molecular Function and

ss, (b) Molecular Function and (c) Cellular Component

Figure S4: GO enrichment analysis. (i) Biological Processes

(a)

enrichment analysis. (i) Biological Processes

(b) (c)

Figure S4b: (ii) Cellular component and (iii) Molecular function

(b) (c)

(iii) Molecular function

Figure S5: Distribution by family in terms of motif nucleotide base composition

Distribution by family in terms of motif nucleotide base composition

Figure S6: Potential codon reiterated in putative TF genes of Tea. (a) Different amino acid pattern, (b) Pie chart representing the abun

amino acids and (c) Graph showing the abundance of amino acids.

Potential codon reiterated in putative TF genes of Tea. (a) Different amino acid pattern, (b) Pie chart representing the abun

undance of amino acids.

Potential codon reiterated in putative TF genes of Tea. (a) Different amino acid pattern, (b) Pie chart representing the abundant

(a) (b)

Figure S7: Amino acid reiterants in proteins (a) Frequency of reiterants of five to ten identical amino acids residues. (

of eleven to twenty identical amino acids residues

(a) (b)

) Frequency of reiterants of five to ten identical amino acids residues. (

of eleven to twenty identical amino acids residues.

) Frequency of reiterants of five to ten identical amino acids residues. (b) Frequency of reiterants

Figure S8a: Codon reiterants (Non-Polar, Aliphatic R Groups

Polar, Aliphatic R Groups)

Figure S8b: Codon reiterants (Aromatic R GroupsAromatic R Groups)

Figure S8c: Codon reiterants (Polar Uncharged R Groups)

Codon reiterants (Polar Uncharged R Groups)

Figure S8d: Codon reiterants (Polar-charged R Groups)

charged R Groups)

(a) (b

Figure S9: SSR frequency decreased as the number of repeat units increased, rate of

(a) (b)

SSR frequency decreased as the number of repeat units increased, rate of decrease was similar in di (a) and tri-

-repeats (b).

Table S4: List of 26 Polymorphic SSR Markers from putative Functional domain of Transcription Factor gene

S.No. Locus Name Repeat Type Size Alleles TF Family Putative Function in Tea Reference

1 TTFMS_13998 (AGG)5 140:240 5 B3 Dormancy status of bud in

Tea

Thirugnanasambantham et al.,

2013

2 TTFMS_9683 (GA)17 250:300 5 BBR-BPC

3 TTFMS_10094 (AT)6 160:200 2 bHLH Secondary metabolism of

Tea

Zhao et al., 2012

4 TTFMS_67364 (TGG)5 170:180 2 bHLH

5 TTFMS_1538_A (CAC)5 150:170 2 bHLH

6 TTFMS_22978 (CT)8 200:600 9 bHLH

7 TTFMS_2467 (TA)6 200:250 2 bHLH

8 TTFMS_17320 (AG)8 150:200 6 C2H2 Dormancy status of bud in

Tea


2013

9 TTFMS_4764 (CAC)5 300:400 1 ERF Response against

abnormal temperature

stress

Wu et al., 2015

10 TTFMS_66493 (GTG)6 150:250 9 FAR1

11 TTFMS_59623 (AG)6 400:500 2 FAR1

12 TTFMS_26427 (TC)8 200:500 11 FAR1

13 TTFMS_36100 (AAG)5 250:400 7 G2-like

14 TTFMS_25851_A (CTG)5 150:200 1 GRAS Response to abiotic

stresses and Gibberelic

acid treatment in tea (Salt

Stress)

Wang et al., 2018

15 TTFMS_299 (CT)9 400:500 3 HSF Regulation under

temperature stress

Liu et al., 2016

16 TTFMS_1264 (TC)8 150:300 5 LBD

17 TTFMS_790 (CT)9 700:800 2 MYB Dormancy and CsMYB4a

is negatively correlated to

accumulation of six

flavan-3-ols and other

phenolic acids

Li et al., 2017 &


2013

18 TTFMS_13383 (AT)6 230:250 1 NAC Accumulate stress-

inducible genes or

metabolites and enhances

stress tolerance (Blister

Blight in Tea)

Wang et al., 2016 & Jayaswall et

al., 2016

19 TTFMS_25118 (TCT)6 150:370 4 Nin-like Bud and Leaf Colour

Development

Wei et al., 2016

20 TTFMS_17254 (CT)8 200:450 4 Nin-like

21 TTFMS_23789 (CAC)6 70:150 4 TCP Leaf Development and Wu et al., 2017

Hormonal stimuli in Tea

22 TTFMS_39438 (CT)6 230-250 2 Trihelix Response to abiotic

stresses in tea (Salt Stress)

Li et al., 2017

23 TTFMS_7051 (CAA)5 250:300 1 Trihelix

24 TTFMS_63882_B (AG)6 150:250 3 Trihelix

25 TTFMS_1910_A (TC)7 220:250 2 WRKY Responses to extreme

temperatures (Cold and

Drought stress) controlling

ABA signalling, Plant

immunity

Jayaswall et al., 2016, Wang et

al., 2016 & Wu et al., 2016 26 TTFMS_42892 (CAA)5 200:300 7 WRKY

(a)

Figure S10: (a) Overall repeat frequency of SSRs in Genome

(b)

eat frequency of SSRs in Genome and (b) Transcriptome data (Transcription Factor (TFs) t

(Transcription Factor (TFs) transcripts) of tea.

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