Informative ISSR Markers Help Identify Genetically Distinct Accessions of Oryza rufipogon in Yield Improvement

doi:10.1016/j.rsci.2016.08.001

Abstract

Abstract:

Inter simple sequence repeat (ISSR) polymorphism was used to determine genetic diversity and phylogenetic relationships in 90 genotypes of wild and cultivated species of Oryza from different geographical regions of the world. In all the 17 primers used in ISSR-PCR, a total of 11 464 bands were amplified at 253 band positions/loci. The primer UBC-809 amplified the maximum bands (1 059) at 21 band positions. UBC-810 and UBC-835 amplified the minimum of 391 bands each at 7 and 14 band positions, respectively. The mean polymorphism information content ranged from 0.44 to 0.84 and resolving power ranged from 8.69 to 23.53. Un-weighted pair group method with arithmetic mean dendrogram and population structure based on the 17 primers separated all genotypes into 4 major clusters with a genetic similarity of 53%-100%. The first two clusters consisted of 30 O. rufipogon accessions each. In the third cluster, O. nivara and O. longistaminata grouped as one sub-cluster and all other O. nivara accessions and cultivars grouped as another sub-cluster. The fourth cluster had only five O. rufipogon accessions which can be a source of new genes. Four sub-populations were identified within O. rufipogon and two sub-populations within O. nivara at K = 7. A subset of six primers with high resolving power values were the most informative and grouped all genotypes almost similarly as the 17 primers did. Use of these six highly informative primers in ISSR-PCR is a cost effective and robust method for assessing genetic diversity in large germplasm collections of wild rice species.

Key words: Oryza, wild species, genetic diversity, population structure, inter simple sequence repeat

Haritha G., Sudhakar T., Chandra D., Ram T., Divya B., Sarla N.. Informative ISSR Markers Help Identify Genetically Distinct Accessions of Oryza rufipogon in Yield Improvement[J]. Rice Science, 2016, 23(5): 225-241.

Figures/Tables 12

Table 1 List of O. rufipogon, O. nivara, O. longistaminata and O. sativa genotypes used and their origins.

N	Acc No.	Species	Source	N	Acc No.	Species	Source	N	Acc No.	Species	Source
1	106147	O. rufipogon	Laos	31	81998	O. rufipogon	Papua new guinea	61	WR119	O. rufipogon	India
2	106277	O. rufipogon	Papua new guinea	32	105125	O. rufipogon	Papua new guinea	62	WR120	O. rufipogon	India
3	106507	O. rufipogon	Myanmar	33	106162	O. rufipogon	Laos	63	81879	O. rufipogon	India
4	106462	O. rufipogon	India	34	81877	O. rufipogon	India	64	81894	O. rufipogon	India
5	106131	O. rufipogon	India	35	81802	O. rufipogon	Indonesia	65	86476	O. rufipogon	India
6	106459	O. rufipogon	India	36	80712	O. rufipogon	India	66	102166	O. nivara	India
7	106427	O. rufipogon	Vietnam	37	81878	O. rufipogon	India	67	105320	O. nivara	India
8	106453	O. rufipogon	Indonesia	38	106274	O. rufipogon	Papua new guinea	68	105333	O. nivara	India
9	106123	O. rufipogon	India	39	104640	O. rufipogon	Vietnam	69	106052	O. nivara	India
10	106437	O. rufipogon	Vietnam	40	81977	O. rufipogon	Indonesia	70	106495	O. nivara	India
11	106338	O. rufipogon	Cambodia	41	104425	O. rufipogon	Thailand	71	105809	O. nivara	Thailand
12	106122	O. rufipogon	India	42	104423	O. rufipogon	Thailand	72	81848	O. nivara	India
13	106271	O. rufipogon	Papua new guinea	43	80669	O. rufipogon	India	73	104721	O. nivara	Thailand
14	81890	O. rufipogon	India	44	80762	O. rufipogon	Myanmar	74	105894	O. nivara	Bangladesh
15	81892	O. rufipogon	India	45	81986	O. rufipogon	Cambodia	75	101230	O. longistaminata	Sierra Leone
16	81894	O. rufipogon	India	46	81987	O. rufipogon	Cambodia	76	101207	O. longistaminata	Ivory coast
17	81884	O. rufipogon	India	47	81989	O. rufipogon	Myanmar	77	IR64	O. sativa	IRRI
18	106465	O. rufipogon	Laos	48	80592	O. rufipogon	India	78	Swarna	O. sativa	India
19	106515	O. rufipogon	Vietnam	49	81880	O. rufipogon	India	79	Rasi	O. sativa	India
20	106119	O. rufipogon	India	50	80781	O. rufipogon	India	80	9314	O. sativa	China
21	106125	O. rufipogon	India	51	81895	O. rufipogon	India	81	BSI115	O. sativa	Indonesia
22	80671	O. rufipogon	India	52	106118	O. rufipogon	India	82	Moroberekan	O. sativa	Guinea
23	106144	O. rufipogon	India	53	106115	O. rufipogon	India	83	Nipponbare	O. sativa	Japan
24	106407	O. rufipogon	Vietnam	54	106084	O. rufipogon	India	84	Taipei 309	O. sativa	Taiwan, China
25	82011	O. rufipogon	India	55	105910	O. rufipogon	Thailand	85	N22	O. sativa	India
26	106163	O. rufipogon	Laos	56	106057	O. rufipogon	India	86	FR13A	O. sativa	India
27	106149	O. rufipogon	Laos	57	104599	O. rufipogon	Sri Lanka	87	Dular	O. sativa	India
28	106340	O. rufipogon	Myanmar	58	104606	O. rufipogon	Sri Lanka	88	Basmati 370	O. sativa	India
29	81978	O. rufipogon	Indonesia	59	106083	O. rufipogon	India	89	Type 3	O. sativa	India
30	81990	O. rufipogon	Myanmar	60	104639	O. rufipogon	Thailand	90	CSR30	O. sativa	India

Table 1 List of O. rufipogon, O. nivara, O. longistaminata and O. sativa genotypes used and their origins.

N	Acc No.	Species	Source	N	Acc No.	Species	Source	N	Acc No.	Species	Source
1	106147	O. rufipogon	Laos	31	81998	O. rufipogon	Papua new guinea	61	WR119	O. rufipogon	India
2	106277	O. rufipogon	Papua new guinea	32	105125	O. rufipogon	Papua new guinea	62	WR120	O. rufipogon	India
3	106507	O. rufipogon	Myanmar	33	106162	O. rufipogon	Laos	63	81879	O. rufipogon	India
4	106462	O. rufipogon	India	34	81877	O. rufipogon	India	64	81894	O. rufipogon	India
5	106131	O. rufipogon	India	35	81802	O. rufipogon	Indonesia	65	86476	O. rufipogon	India
6	106459	O. rufipogon	India	36	80712	O. rufipogon	India	66	102166	O. nivara	India
7	106427	O. rufipogon	Vietnam	37	81878	O. rufipogon	India	67	105320	O. nivara	India
8	106453	O. rufipogon	Indonesia	38	106274	O. rufipogon	Papua new guinea	68	105333	O. nivara	India
9	106123	O. rufipogon	India	39	104640	O. rufipogon	Vietnam	69	106052	O. nivara	India
10	106437	O. rufipogon	Vietnam	40	81977	O. rufipogon	Indonesia	70	106495	O. nivara	India
11	106338	O. rufipogon	Cambodia	41	104425	O. rufipogon	Thailand	71	105809	O. nivara	Thailand
12	106122	O. rufipogon	India	42	104423	O. rufipogon	Thailand	72	81848	O. nivara	India
13	106271	O. rufipogon	Papua new guinea	43	80669	O. rufipogon	India	73	104721	O. nivara	Thailand
14	81890	O. rufipogon	India	44	80762	O. rufipogon	Myanmar	74	105894	O. nivara	Bangladesh
15	81892	O. rufipogon	India	45	81986	O. rufipogon	Cambodia	75	101230	O. longistaminata	Sierra Leone
16	81894	O. rufipogon	India	46	81987	O. rufipogon	Cambodia	76	101207	O. longistaminata	Ivory coast
17	81884	O. rufipogon	India	47	81989	O. rufipogon	Myanmar	77	IR64	O. sativa	IRRI
18	106465	O. rufipogon	Laos	48	80592	O. rufipogon	India	78	Swarna	O. sativa	India
19	106515	O. rufipogon	Vietnam	49	81880	O. rufipogon	India	79	Rasi	O. sativa	India
20	106119	O. rufipogon	India	50	80781	O. rufipogon	India	80	9314	O. sativa	China
21	106125	O. rufipogon	India	51	81895	O. rufipogon	India	81	BSI115	O. sativa	Indonesia
22	80671	O. rufipogon	India	52	106118	O. rufipogon	India	82	Moroberekan	O. sativa	Guinea
23	106144	O. rufipogon	India	53	106115	O. rufipogon	India	83	Nipponbare	O. sativa	Japan
24	106407	O. rufipogon	Vietnam	54	106084	O. rufipogon	India	84	Taipei 309	O. sativa	Taiwan, China
25	82011	O. rufipogon	India	55	105910	O. rufipogon	Thailand	85	N22	O. sativa	India
26	106163	O. rufipogon	Laos	56	106057	O. rufipogon	India	86	FR13A	O. sativa	India
27	106149	O. rufipogon	Laos	57	104599	O. rufipogon	Sri Lanka	87	Dular	O. sativa	India
28	106340	O. rufipogon	Myanmar	58	104606	O. rufipogon	Sri Lanka	88	Basmati 370	O. sativa	India
29	81978	O. rufipogon	Indonesia	59	106083	O. rufipogon	India	89	Type 3	O. sativa	India
30	81990	O. rufipogon	Myanmar	60	104639	O. rufipogon	Thailand	90	CSR30	O. sativa	India

Table 2 Details of 17 primers used in ISSR-PCR.

Primer	Sequence repeat motif (5′-3′)	Annealing temperature (°C)
UBC-807	(AG)₈T	47
UBC-808	(AG)₈C	49
UBC-809	(AG)₈G	49
UBC-810	(GA)₈T	47
UBC-811	(GA)₈C	47
UBC-812	(GA)₈A	47
UBC-823	(TC)₈C	49
UBC-824	(GA)₈G	49
UBC-834	(GA)₈YT	48
UBC-835	(AG)₈TC	50
UBC-840	(GA)₈YT	47
UBC-842	(GA)₈YG	50
UBC-853	(TC)₈RT	48
UBC-854	(TC)₈RG	48
UBC-884A	HBH(AG)₇	47
UBC-884B	ACTGCT(GA)₇	50
UBC-885	GCCG(AG)₆	50

Fig. 1. Fingerprints of 90 samples using primer UBC-823 by inter simple sequence repeat markers. M, 100-bp ladder. 1 to 90 are the numbers of the genotypes which are shown in Table 1.

Table 3 Details for primers.

Primer	PIC	Resolving power (Rp)	No. of band positions	Total No. of bands amplified	Range of bands amplified by primer ^b
UBC-807	0.84	14.11	19	635	11-53
UBC-808	0.7	13.27	13	597	12-67
UBC-809 ^a	0.65	23.53	21	1059	23-83
UBC-810	0.59	8.69	7	391	41-76
UBC-811	0.44	10.36	7	466	52-86
UBC-812 ^a	0.64	19.98	18	899	18-84
UBC-823	0.73	14.58	15	656	23-79
UBC-824	0.72	16.27	16	732	26-70
UBC-834 ^a	0.69	17.09	17	769	15-81
UBC-835	0.84	8.69	14	391	4-81
UBC-840 ^a	0.67	18.04	17	812	12-70
UBC-842 ^a	0.61	18.6	16	837	11-86
UBC-853	0.84	12.93	18	582	4-59
UBC-854	0.64	14.93	13	672	22-85
UBC-884A	0.62	16.18	14	728	17-80
UBC-884B	0.79	9.51	12	428	5-82
UBC-885 ^a	0.64	18	16	810	21-80

Fig. 2. Frequency distribution of polymorphic bands across 90 accessions.

Fig. 3. Polymorphism information content (PIC) values for the polymorphic bands obtained using ISSR primers in 90 accessions.

Fig. 4. Un-weighted pair group method with arithmetic mean dendrogram among 90 accessions using 17 primers. r, O. rufipogon; n, O. nivara; l, O. longistaminata; s, O. sativa. Accession numbers (from top to bottom) are 1, 23, 3, 28, 29, 24, 26, 30, 21, 22, 25, 27, 2, 13, 11, 14, 15, 16, 17, 6, 7, 10, 8, 4, 18, 9, 12, 5, 20, 19, 31, 47, 37, 33, 34, 35, 54, 51, 50, 55, 56, 57, 58, 48, 49, 38, 39, 40, 42, 43, 44, 45, 46, 36, 41, 60, 52, 53, 32, 59, 71, 72, 73, 74, 75, 76, 66, 67, 69, 68, 70, 77, 78, 80, 81, 82, 83, 84, 88, 89, 85, 86, 87, 79, 90, 61, 62, 65, 63 and 64.

Fig. 5. Un-weighted pair group method with arithmetic mean dendrogram among 90 accessions using 6 informative primers. r, O. rufipogon; n, O. nivara; l, O. longistaminata; s, O. sativa. Accession numbers (from top to bottom) are 1, 23, 27, 2, 13, 11, 4, 18, 21, 6, 8, 7, 10, 9, 12, 14, 15, 16, 22, 20, 24, 30, 28, 29, 26, 57, 17, 25, 5, 32, 3, 58, 19, 36, 41, 60, 52, 53, 38, 31, 49, 33, 34, 35, 51, 37, 54, 48, 50, 39, 40, 42, 43, 44, 45, 46, 47, 55, 56, 71, 74, 75, 76, 72, 73, 59, 66, 67, 69, 68, 77, 78, 85, 80, 70, 81, 82, 83, 84, 86, 87, 79, 90, 88, 89, 61, 63, 64, 62 and 65.

Fig. 6. Two dimensional plots of the genetic relationship among 90 genotypes of Oryza as revealed by principal coordinate analysis (PCoA) in NTSys ver. 2.02. The five O. rufipogon accessions 61, 62, 63, 64 and 65 separated from the other O. rufipogon accessions and grouped together.

Fig. 7. An unrooted neighbor joining tree showing the genetic relationships among the 90 genotypes. Red indicates 30 O. rufipogon accessions of subgroup I, blue indicates another 30 O. rufipogon accessions of subgroup II, and yellow indicates O. nivara, O. longistaminata and O. sativa accessions. The five distinct O. rufipogon accessions of subgroup III are in green color.

Fig. 8. Factorial correspondence analysis of 90 genotypes using 17 primers. Red indicates O. rufipogon accessions of sub-group I, blue indicates O. rufipogon accessions of sub-group II and yellow indicates O. nivara, O. longistaminata and O. sativa accessions. The five distinct O. rufipogon accessions of sub-group III are in green color.

Fig. 9. STRUCTURE analysis of the 90 accessions. A, The graphical representation showing population structure of two peaks. One peak at K = 4 showing largely inter-species differentiation and the other peak at K = 7 showing largely intra-species differentiation within wild and cultivated genotypes. B, A model-based clustering method at K = 4 (the first panel). Each color represents different population groups. I, II, and III represent sub-populations within O. rufipogon; IV represents O. nivara, O. longistaminata and O. sativa. C, A model-based clustering method at K = 7 (the second panel). Each color represents different population groups. I, II, III and IV represent four sub-populations within O. rufipogon; V and VI represent sub-populations within O. nivara accessions and two O. longistaminata also grouped here; VII represents population consisting of all the five sub-groups of O. sativa. A, The graphical representation showing population structure of two peaks. One peak at K = 4 showing largely inter-species differentiation and the other peak at K = 7 showing largely intra-species differentiation within wild and cultivated genotypes. B, A model-based clustering method at K = 4 (the first panel). Each color represents different population groups. I, II, and III represent sub-populations within O. rufipogon; IV represents O. nivara, O. longistaminata and O. sativa. C, A model-based clustering method at K = 7 (the second panel). Each color represents different population groups. I, II, III and IV represent four sub-populations within O. rufipogon; V and VI represent sub-populations within O. nivara accessions and two O. longistaminata also grouped here; VII represents population consisting of all the five sub-groups of O. sativa.

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