Development of Novel Genomic Simple Sequence Repeat (g-SSR) Markers and Their Validation for Genetic Diversity Analyses in Kalmegh [Andrographis paniculata (Burm. F.) Nees]
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. Plant Genomic DNA (gDNA) Isolation
2.3. Construction of the Microsatellite-Enriched Library
2.4. SSR Finding and Primer Designing
2.5. Polymerase Chain Reaction
2.6. Data Scoring and Statistical Analyses
3. Results and Discussion
3.1. Development of SSR Markers from Enriched Genomic Libraries
3.2. Validation of g-SSR Loci and Genetic Diversity Statistics
3.3. Cluster Analysis
3.4. Population Structure
3.5. AMOVA, PCoA, and Mantel Test
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sl.No. | Accession No. | Habitat | Collection Site | Collection Year | Agro Ecological Regions |
---|---|---|---|---|---|
1 | IC 111286 | Disturbed | Haidargarh, Faizabad, Uttar Pradesh | 1992 | Gangatic Plains |
2 | IC 111287 | Disturbed | Katarnia Forest, Gonda, Uttar Pradesh | 1992 | Gangatic Plains |
3 | IC 111288 | Natural | BastiForest, Basti, Uttar Pradesh | 1992 | Gangatic Plains |
4 | IC 111290 | Cultivated | Regional Research Lab (RRL), Jorhat, Assam | 1992 | N.E. Region |
5 | IC 111291 | Disturbed | Shola Forest, Thiruananthapuram, Kerala | 1992 | Western Ghats |
6 | IC 210635 | Cultivated | Garden, Namakkal, Tamil Nadu | 1997 | Eastern Ghats |
7 | IC 211295 | Natural | Ananthagiri Forest, Visakhapatnam, Andhra Pradesh | 1997 | Eastern Ghats |
8 | IC 333252 | Disturbed | Jamli, Barwani, Madhya Pradesh | 1999 | Eastern Ghats |
9 | IC 342134 | Natural | Suparia Forest, Bahraich, Uttar Pradesh | 1995 | Gangatic Plains |
10 | IC 342135 | Natural | Satrikh Forest, Barbanki, Uttar Pradesh | 1995 | Gangatic Plains |
11 | IC 342136 | Cultivated | Garden, Saharanpur, Uttar Pradesh | 1994 | Gangatic Plains |
12 | IC342137 | Cultivated | Regional Research Centre (RRC), Pune, Maharashtra | 1995 | Western Ghats |
13 | IC 342138 | Cultivated | Regional Research Centre (RRC), Thiruananthapuram, Kerala | 1995 | Western Ghats |
14 | IC 342139 | Cultivated | Regional Research Centre (RRC), Jhansi, Uttar Pradesh | 1995 | Gangatic Plains |
15 | IC 342140 | Cultivated | Garden, RRC-Kolkata, West Bangal | 1995 | N. E. Region |
16 | IC 342141 | Cultivated | Narendra Dev University, Faizabad, Uttar Pradesh | 1995 | Gangatic Plains |
17 | IC 399612 | Natural | Kashidih Forest, East Singhbhum, Jharkhand | 1999 | Gangatic Plains |
18 | IC 400519 | Cultivated | Adumalleshwaram, Chitradurg, Karnataka | 2001 | Western Ghats |
19 | IC 437223 | Cultivated | YS Parmar University, Solan, Himachal Pradesh | 2003 | Western Himalaya |
20 | IC 471889 | Natural | Dindori Forest, Amarkantak, Madhya Pradesh | 2003 | Eastern Ghats |
21 | IC 471890 | Natural | Dindori Forest, Amarkantak, Madhya Pradesh | 2003 | Eastern Ghats |
22 | IC 471891 | Natural | Dindori Forest, Amarkantak, Madhya Pradesh | 2003 | Eastern Ghats |
23 | IC 471892 | Natural | Dindori Forest, Amarkantak, Madhya Pradesh | 2003 | Eastern Ghats |
24 | IC 471893 | Natural | Dindori Forest, Amarkantak, Madhya Pradesh | 2003 | Eastern Ghats |
25 | IC 471894 | Natural | Dindori Forest, Amarkantak, Madhya Pradesh | 2003 | Eastern Ghats |
26 | IC 471895 | Natural | Dindori Forest, Amarkantak, Madhya Pradesh | 2003 | Eastern Ghats |
27 | IC 471912 | Disturbed | Nauni Forest, Solan, Himachal Pradesh | 2002 | Western Himalaya |
28 | IC 471913 | Disturbed | Nauni Forest, Solan, Himachal Pradesh | 2002 | Western Himalaya |
29 | IC 471914 | Disturbed | Ochghat, Solan, Himachal Pradesh | 2002 | Western Himalaya |
30 | IC 471915 | Disturbed | BatalGhatti, Solan, Himachal Pradesh | 2002 | Western Himalaya |
31 | IC 471916 | Disturbed | Ochghat, Solan, Himachal Pradesh | 2002 | Western Himalaya |
32 | IC 471917 | Disturbed | Kunihar, Solan, Himachal Pradesh | 2002 | Western Himalaya |
33 | IC 471918 | Disturbed | BatalGhatti, Solan, Himachal Pradesh | 2002 | Western Himalaya |
34 | IC 471919 | Disturbed | BatalGhatti, Solan, Himachal Pradesh | 2002 | Western Himalaya |
35 | IC 471896 | Natural | Mandla Forest, Mandla, Madhya Pradesh | 2003 | Eastern Ghats |
36 | IC 421397 | Natural | Machkot, Bastar, Chhattisgarh | 2001 | Eastern Ghats |
37 | IC 421431 | Natural | Barasur Forest, Dantewada, Chhattisgarh | 2001 | Eastern Ghats |
38 | IC 421432 | Natural | Barasur Forest, Dantewada, Chhattisgarh | 2001 | Eastern Ghats |
39 | IC 421435 | Natural | Greedam, Dantewada, Chhattisgarh | 2001 | Eastern Ghats |
40 | IC 421436 | Natural | Muchnar, Dantewada, Chhattisgarh | 2001 | Eastern Ghats |
41 | IC 421442 | Natural | Dantewara Forest, Dantewada, Chhattisgarh | 2001 | Eastern Ghats |
42 | IC 264272 | Natural | Ishapur, Dalsinghsarai, Bihar | 2001 | Gangatic Plains |
S.No | Primer ID | Forward Primer Sequence (5′-3′) | Reverse Primer Sequence (5′-3′) | Repeat Motif | Expected Product Size (bp) |
---|---|---|---|---|---|
1 | Ando4-2 | CTCTCTCTCGCAGCTCTCTCTC | CTTCGGATCAGTTAGCCCCT | (CT)7 | 386 |
2 | Ando6-3 | AGCTTCGGATCAGTTAGTCCCT | GCTCCCTCTCAGTGTCTCTCTC | (AG)6 | 378 |
3 | Ando2-3-2 | GCTTCGGATCAGTTAGTCCCTT | AGCTCTCTCTCGCAGCTCTCT | (AG)6 | 388 |
4 | Ando2-3-3 | GAGAGACCTGCGAGAGAGAGAG | GAACCAGGCAGAACCAATAATC | (GA)6 | 241 |
5 | Ando2-6-2 | GCTTCGGATCAGTTAGTCCCTT | CTCTCTCTCGCAGCTCTCTCTC | (GA)7 | 387 |
6 | Ando2-9 | GATTATGTGGGAATCTTGGGTG | ATATAGGTGGGCGATAAACCG | (A)15 | 228 |
7 | Ando2-12 | AACAAGGTTACACTCTCCGACC | CTCGATCCTATTCAGTTCCACC | (A)13 | 388 |
8 | Ando2-23 | TTCTTTTCTGTGTAATCGTCGC | CTAAGCGTTGCTCCATTTCTTC | (A)10 | 188 |
9 | Ando2-24-3 | CGGCTCTCTCTCAGTCTCTCTC | CTTCGGGTCAGTTAGTCCCTT | (CT)7 | 375 |
10 | Ando2-30-2 | AGCTTCGGATCAGTTAGTCCCT | CGCTCGTAGTCTCTCTCTCACA | (AG)6 | 258 |
11 | Ando2-31-2 | ATTGATGCCCAAAGAGAAGAAG | CTCTCCCTATCTCGCACTATCG | (AG)6 | 250 |
12 | Ando2-32-2 | AGCTTCGGATCAGTTAGTCCCT | CAGCTCTCCCTCAGTCTCTCTC | (AG)6 | 378 |
13 | Ando2-40-2 | CTCTCTCTCTCTCTCCACAGCC | ATGACCCTCAACATAGCGTTTT | (TC)18 | 316 |
14 | Ando 4-2-1 | ATGACCCTCAACATAGCGTTTT | CTCTCTCTCTCTCTCCACAGCC | (GA)13 | 318 |
15 | Ando4-3-2 | AGCTTCGGATCTCTCTCCACT | ATGACCCTCAACATAGCGTTTT | (TC)9 | 383 |
16 | Ando4-4-2 | CTTCGGGTCAGTTAGTCCCTT | CAGCTCCCTCTCAGTCTCTCTC | (AG)6 | 374 |
17 | Ando4-11-2 | AGCTTCGGATCTCTCTCCACT | ATGACCCTCAACATAGCGTTTT | (TC)9 | 379 |
18 | Ando2-3 | AGCTTCGGATCAGTTAGTCCCT | TCTCTATCTCGCATTCTCTCCC | (AG)6 | 478 |
19 | Ando2-21 | GCCCAAAGAGAAATAGCTGAGA | CTATGACCATGATTACGCCAAG | (GA)6 | 298 |
20 | Ando2-30-1 | GCTTCGGATCAGTTAGTCCCTT | CGCAGCTCTCTCTCAGTCTCTC | (AG)6 | 381 |
21 | Ando2-30-3 | AGCTTCGGATCAGTTAGTCCCT | TATCTCGCACTCTCTCTCTGGC | (GA)7 | 479 |
22 | Ando2-31-1 | CTTCGGATCGGTTAGTCCCT | CTCTCTCTCGCAGCTCTCTCTC | (AG)6 | 390 |
23 | Ando2-31-3 | CTTCGGATCGGTTAGTCCCT | TATCTCGCACTCTCTCTCTGGC | (GA)7 | 479 |
24 | Ando4-4-1 | CTTCGGGTCAGTTAGTCCCTT | CAGCTCCCTCTCAGTCTCTCTC | (AG)6 | 374 |
25 | Ando4-4-3 | CTTCGGGTCAGTTAGTCCCTT | TCTCTATCTCGCACTCTCTCCC | (GA)7 | 477 |
26 | Ando4-9 | CCAGTCCTTTTCTGCTGTTACC | AGCTTCGATCAATTTCCAAGG | (AG)10 | 173 |
27 | Ando4-9-2 | GCTTCGGATCAAAATACTCAGC | CTCTCTTTATGGCCTATCCCCT | (AGGGAG)5 | 298 |
28 | Ando4-21 | AGCTTCGGATCTCTCTCCACT | ATGACCCTCAACATAGCGTTTT | (TC)12 | 383 |
29 | Ando4-26 | AGCTTCGGATCGTAGGGTTT | TCTGTATGTGTGCTCAACCTCC | (TC)14 | 235 |
30 | Ando4-27 | ATGACCCTCAACATAGCGTTTT | CTCTCTCTCTCTCTCCACAGCC | (GA)19 | 318 |
31 | Ando4-27-2 | ATGACCCTCAACATAGCGTTTT | AGCTTCGGATCTCTCTCCACT | (GA)13 | 385 |
32 | Ando4-31 | AGCTTCGGATCAGTTAGTCCCT | TTTCCCTCTCTATCTCGCACTC | (AG)6 | 489 |
33 | Ando4-32 | AGCTTCGGATCAGTTAGTCCCT | GCAGTCTCTCTCGCAACTCTCT | (AG)6 | 449 |
34 | Ando4-32-1 | AGCTTCGGATCAGTTAGTCCCT | TGCAGCTCTCTCTCTCTCAGTTT | (GA)6 | 499 |
35 | Ando4-34-1 | CTCTCTCTCTCTCTCCACAGCC | CAACCTCCATCATCTGAACAAA | (TC)18 | 253 |
36 | Ando4-34-2 | AGCTTCGGATCTCTCTCCACT | ATGACCCTCAACATAGCGTTTT | (TC)12 | 385 |
37 | Ando4-35-1 | CAACCTCCATCATCTGAACAAA | CTCTCTCTCTCTCTCCACAGCC | (GA)19 | 251 |
38 | Ando4-35-2 | ATGACCCTCAACATAGCGTTTT | AGCTTCGGATCTCTCTCCACT | (GA)13 | 385 |
39 | Ando4-36 | AGCTTCGGATCAGTTAGTCCCT | TATCTCGCACTCTCTCTCTGGC | (GA)6 | 471 |
40 | Ando4-36-2 | AGCGATAGTGCGTGATAGGG | GGCCTCTCTCAGTTACAGTCTCC | (GA)6 | 276 |
41 | Ando4-39 | AGCTTCGGATCGTAGGGTTT | TCTGTATGTGTGCTCAACCTCC | (TC)13 | 233 |
42 | Ando4-41 | CTTCGGGTCAGTTAGTCCCTT | TCTCTATCTCGCACTCTCTCCC | (GA)7 | 479 |
43 | Ando4-42 | AATTCCCACAGCAGAGAGAGAG | GTTTCTGACTTTTCACGTTCCC | (GA)14 | 331 |
44 | Ando4-43/1 | CTCTCTCTCTCTCTCCACAGCC | TGACCCTCAACATAGCGTCTTA | (TC)19 | 317 |
45 | Ando4-43/2 | AGCTTCGGATCTCTCTCCACT | TGACCCTCAACATAGCGTCTTA | (TC)12 | 382 |
46 | Ando5-1 | TAACCGAGCATCTCTCTCTGCT | TCAATGGGTATCTGTGTTTTGG | (TCT)4 | 120 |
47 | Ando5-8 | GCTTCGGATCTAACACAACCTC | GAAAAGGGTTCTCCTCCAGTTT | (TCTT)3 | 187 |
48 | Ando5-10 | TTGATGCCCAAAGAGAAATAGC | GTTACAGTCTCCCTTGCAGCTC | (AG)6 | 487 |
49 | Ando5-12 | GAGCGATAGTGCGAGATAGGG | GTTACAGTCTCCCTTGCAGCTC | (GA)8 | 270 |
50 | Ando5-12-1 | CTTCGGATCAGTTAGCCCCT | GTCTTGCACCCACTCTCTCTCT | (GA)6 | 319 |
51 | Ando5-13 | CTCTCTCTCTCTCTCCACAGCC | AAGCGGGATTGATTTACAACAC | (TC)15 | 388 |
52 | Ando5-13-2 | AGCTTCGGATCTCTCTCCACT | AAGCGGGATTGATTTACAACAC | (TC)15 | 459 |
53 | Ando5-14 | AGCTTCGGATCAGTTAGTCCCT | TATCTCGCACTCTCTCTCTGGC | (GA)7 | 473 |
54 | Ando5-14-2 | AGCTTCGGATCAGTTAGTCCCT | CGCACTCTCTCAGTTTTCCTCT | (AG)6 | 345 |
55 | Ando5-19 | GAAGACCCTAATCGAAACATCG | AAAGAACCTCCGCTCATAACAG | (TCTTC)2 | 264 |
56 | Ando5-23 | AGCTTCGGATCAGTTAGTCCCT | GCTCTCTCTCTCGCAGTTTCTC | (AG)6 | 500 |
57 | Ando5-26 | ATTCGGTCATTCTTAGCCCTCT | TCAATGGGTATCTGTGTTTTGG | (TCT)4 | 158 |
58 | Ando5-26-2 | ACCGAGCATCTCTCTCTGCTAT | TTCGGATCTGTCCTGTGTTTC | (AACTC)2 | 224 |
59 | Ando5-29 | CTTCGGATCAGTTAGTCCCTTC | TCTCTATCTCGCAGCTCTCCTT | (GA)6 | 413 |
60 | Ando5-29-2 | AGCTTCGGATCAGTTAGTCCCT | TCTCTCTCTCCCTATCTCGCAC | (AG)6 | 281 |
61 | Ando5-30 | GACAACACATTCCTCAAAAGCC | AGCTTCGGATCTGGTCTAACG | (TC)8 | 141 |
62 | Ando5-31 | CTTCGGGTCAGTTAGTCCCTT | TCCCTCTCTATCTCGCACTCTC | (GA)6 | 481 |
63 | Ando5-31-2 | CGAGCGATAGTGCGTGATA | TCTCCCTCTCCCAGTCTCTC | (GA)6 | 324 |
64 | Ando5-36 | CTTCGGGTCAGTTAGTCCCTT | TCTCTCTCGCAGGTCTCTCTCT | (GA)7 | 325 |
65 | Ando5-37 | CTCCTTGACTATCTTTGGCCTG | TTATGTCTCTGATGATGGGTCG | (TCTTC)2 | 136 |
66 | Ando5-38 | CTCTCTCTCTCTCTCCACAGCC | ATGACCCTCAACATAGCGTTTT | (TC)19 | 318 |
67 | Ando5-38-2 | AGCTTCGGATCTCTCTCCACT | ATGACCCTCAACATAGCGTTTT | (TC)14 | 387 |
Sl.No. | Primer ID | Ta x(°C) | Allele Size Range (bp) | Allele No | Major Allele Frequency | Gene Diversity (Expected Heterozygosity) | Observed Heterozygosity | PIC y |
---|---|---|---|---|---|---|---|---|
1. | Ando 4-2 | 45.0 | 160–170 | 2 | 0.81 | 0.31 | 0 | 0.26 |
2. | Ando 2-24-3 | 65.4 | 360–400 | 4 | 0.71 | 0.41 | 0 | 0.33 |
3. | Ando 2-30-2 | 59.9 | 270–300 | 4 | 0.63 | 0.45 | 0 | 0.35 |
4. | Ando 4-4-2 | 60.9 | 370–390 | 2 | 0.76 | 0.36 | 0 | 0.30 |
5. | Ando 4-3-2 | 60.9 | 390–490 | 6 | 0.75 | 0.35 | 0 | 0.28 |
6. | Ando 4-11-2 | 60.9 | 390–410 | 2 | 0.85 | 0.26 | 0 | 0.22 |
7. | Ando 4-2-1 | 60.9 | 320–360 | 4 | 0.70 | 0.41 | 0.19 | 0.33 |
8. | Ando 4-40-2 | 62.0 | 310–320 | 2 | 0.71 | 0.42 | 0 | 0.33 |
9. | Ando 2-32-2 | 40.9 | 380–390 | 2 | 0.68 | 0.44 | 0 | 0.34 |
10. | Ando 2-31-2 | 40.9 | 250–260 | 2 | 0.53 | 0.50 | 0 | 0.37 |
11. | Ando 2-21 | 41.9 | 720-760 | 2 | 0.89 | 0.19 | 0 | 0.17 |
12. | Ando 4-9-2 | 40.9 | 780–820 | 4 | 0.70 | 0.42 | 0 | 0.33 |
13. | Ando 4-21 | 59.3 | 410–820 | 4 | 0.72 | 0.41 | 0 | 0.32 |
14. | Ando 4-26 | 50.9 | 240–250 | 2 | 0.54 | 0.50 | 0 | 0.37 |
15. | Ando 4-27 | 45.6 | 310–340 | 4 | 0.84 | 0.25 | 0 | 0.21 |
16. | Ando 4-27-2 | 43.0 | 390–400 | 2 | 0.52 | 0.50 | 0 | 0.37 |
17. | Ando 4-31 | 40.9 | 290–360 | 8 | 0.66 | 0.41 | 0 | 0.32 |
18. | Ando 4-32 | 40.4 | 210–360 | 6 | 0.72 | 0.39 | 0 | 0.31 |
19. | Ando 4-32-1 | 48.4 | 360–450 | 8 | 0.73 | 0.36 | 0 | 0.29 |
20. | Ando 4-34-1 | 54.3 | 310–320 | 2 | 0.69 | 0.43 | 0 | 0.34 |
21. | Ando 4-34-2 | 54.3 | 320–430 | 8 | 0.69 | 0.40 | 0 | 0.31 |
22. | Ando 4-35-1 | 54.3 | 220–280 | 4 | 0.61 | 0.47 | 0.15 | 0.36 |
23. | Ando 4-35-2 | 43.0 | 320–410 | 6 | 0.64 | 0.45 | 0 | 0.35 |
24. | Ando 4-36-2 | 43.0 | 230–340 | 4 | 0.95 | 0.10 | 0 | 0.09 |
25. | Ando 4-39 | 40.9 | 140-260 | 4 | 0.77 | 0.36 | 0.06 | 0.29 |
26. | Ando 4-41 | 40.9 | 620–680 | 6 | 0.66 | 0.39 | 0 | 0.30 |
27. | Ando 4-43/1 | 40.4 | 320–350 | 4 | 0.58 | 0.49 | 0 | 0.37 |
28. | Ando 4-43/2 | 40.9 | 390–430 | 4 | 0.68 | 0.41 | 0 | 0.32 |
29. | Ando 5-1 | 51.9 | 420–450 | 4 | 0.64 | 0.45 | 0 | 0.35 |
30. | Ando 5-10 | 40.1 | 260–490 | 4 | 0.66 | 0.44 | 0.10 | 0.35 |
31. | Ando 5-12 | 49.3 | 100–220 | 8 | 0.82 | 0.30 | 0 | 0.25 |
32. | Ando 5-12-1 | 51.9 | 370–560 | 4 | 0.54 | 0.49 | 0.21 | 0.37 |
33. | Ando 5-13 | 58.4 | 390–420 | 4 | 0.76 | 0.37 | 0 | 0.30 |
34. | Ando 5-13-2 | 51.9 | 390–510 | 4 | 0.64 | 0.44 | 0 | 0.34 |
35. | Ando 5.14 | 51.9 | 330–360 | 4 | 0.66 | 0.43 | 0 | 0.34 |
36. | Ando 5-14-2 | 40.4 | 390–410 | 2 | 0.52 | 0.50 | 0 | 0.37 |
37. | Ando 5-26-2 | 54.3 | 850–870 | 2 | 0.53 | 0.50 | 0 | 0.37 |
38. | Ando 5-29 | 54.3 | 420–440 | 2 | 0.50 | 0.50 | 0 | 0.38 |
39. | Ando 5-30 | 40.4 | 250–350 | 4 | 0.65 | 0.41 | 0 | 0.32 |
40. | Ando 5-31-2 | 40.4 | 330–550 | 4 | 0.65 | 0.45 | 0.12 | 0.35 |
41. | Ando 5-37 | 51.9 | 210–290 | 4 | 0.70 | 0.41 | 0 | 0.33 |
Mean | 3.95 | 0.68 | 0.40 | 0.02 | 0.32 |
Source | df | SS | MS | Estimated Variance | % |
---|---|---|---|---|---|
Among Populations | 2 | 310.537 | 155.268 | 4.453 | 22% |
Among Individual | 39 | 1250.952 | 32.076 | 15.877 | 77% |
Within Individual | 42 | 13.5 | 0.321 | 0.321 | 1% |
Total | 83 | 1574.988 | 20.651 | 100% |
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Kumar, R.; Kumar, C.; Paliwal, R.; Roy Choudhury, D.; Singh, I.; Kumar, A.; Kumari, A.; Singh, R. Development of Novel Genomic Simple Sequence Repeat (g-SSR) Markers and Their Validation for Genetic Diversity Analyses in Kalmegh [Andrographis paniculata (Burm. F.) Nees]. Plants 2020, 9, 1734. https://doi.org/10.3390/plants9121734
Kumar R, Kumar C, Paliwal R, Roy Choudhury D, Singh I, Kumar A, Kumari A, Singh R. Development of Novel Genomic Simple Sequence Repeat (g-SSR) Markers and Their Validation for Genetic Diversity Analyses in Kalmegh [Andrographis paniculata (Burm. F.) Nees]. Plants. 2020; 9(12):1734. https://doi.org/10.3390/plants9121734
Chicago/Turabian StyleKumar, Ramesh, Chavlesh Kumar, Ritu Paliwal, Debjani Roy Choudhury, Isha Singh, Ashok Kumar, Abha Kumari, and Rakesh Singh. 2020. "Development of Novel Genomic Simple Sequence Repeat (g-SSR) Markers and Their Validation for Genetic Diversity Analyses in Kalmegh [Andrographis paniculata (Burm. F.) Nees]" Plants 9, no. 12: 1734. https://doi.org/10.3390/plants9121734