Morphological description, genetic diversity and population structure of safflower (Carthamus tinctorius L.) mini core collection using SRAP and SSR markers

Figures & data

Table 1. Details of 135 safflower accessions (Accession name and origin) subjected for genetic diversity analysis.

No.	Accession	Origin	No.	Accession	Origin	No.	Accession	Origin
1	PI388909	Iran	46	PI340096	Turkey	91	PI288307	India
2	PI388908	Iran	47	PI304454	Iran	92	PI306903	India
3	PI304472	Iran	48	PI237542	Sudan	93	PI248377	India
4	PI307070	India	49	PI304453	Iran	94	PI560170	United States
5	PI307074	India	50	PI426189	Afghanistan	95	PI248795	India
6	PI307073	India	51	PI304500	Turkey	96	PI544016	China
7	PI307072	India	52	PI237546	Sudan	97	PI304443	Iran
8	PI525458	United States	53	PI258421	Portugal	98	PI426186	Afghanistan
9	PI251980	Turkey	54	PI406016	Iran	99	PI283759	India
10	PI406000	Iran	55	PI304452	Iran	100	PI426517	Pakistan
11	PI307068	India	56	PI304451	Iran	101	PI405969	Iran
12	PI613425	United States	57	PI292000	Israel	102	PI304438	Iran
13	PI451957	India	58	PI304508	Turkey	103	PI250598	Pakistan
14	PI451958	India	59	PI304471	Iran	104	PI304408	Pakistan
15	PI537688	United States	60	PI304458	Iran	105	PI301055	Turkey
16	PI306900	India	61	PI568822	China	106	PI388903	Iran
17	PI250078	Egypt	62	PI527710	United States	107	PI251462	Turkey
18	PI304463	Iran	63	PI537631	United States	108	PI226993	Israel
19	PI250205	Pakistan	64	PI537628	United States	109	PI306898	India
20	PI304591	Afghanistan	65	PI405966	Iran	110	PI537629	United States
21	PI248837	India	66	PI253515	Germany	111	PI304453	Iran
22	PI304592	Afghanistan	67	PI283754	India	112	PI258417	Portugal
23	PI572419	United States	68	PI283755	India	113	PI258418	Portugal
24	PI560202	United States	69	PI304474	Iran	114	PI392031	Turkey
25	PI560201	United States	70	PI306924	India	115	PI248794	India
26	PI613529	China	71	PI514630	China	116	PI405998	Iran
27	PI237552	Morocco	72	PI248840	India	117	PI406003	Iran
28	PI304467	Iran	73	PI568821	China	118	PI470942	Bangladesh
29	PI304466	Iran	74	PI248799	India	119	PI527710	Mexico
30	PI305178	India	75	PI177302	Turkey	120	PI248381	India
31	PI253537	China	76	PI183669	Turkey	121	PI613526	Turkey
32	PI537671	United States	77	PI537615	United States	122	PI304465	Iran
33	PI406004	Iran	78	PI506426	China	123	R472	Iran
34	PI304449	Iran	79	PI304473	Iran	124	RLR755/292	Iran
35	PI305168	India	80	PI426515	Pakistan	125	6LR/55/65 varamin	Iran
36	PI305163	India	81	PI258416	Portugal	126	5LR51/20/6varamin	Iran
37	PI305161	India	82	PI544011	China	127	R25	Iran
38	PI283773	India	83	PI248839	India	128	415	Iran
39	PI572414	United States	84	PI251289	Israel	129	17	Iran
40	PI250082	Egypt	85	PI251290	Israel	130	SNC/1	Iran
41	PI406000	Iran	86	PI537683	United States	131	9	Iran
42	PI544032	China	87	PI304502	Turkey	132	Sina	Iran
43	PI239043	Morocco	88	PI306995	India	133	LRV	Iran
44	PI537621	United States	89	PI283777	India	134	Faraman	Iran
45	PI544030	China	90	PI392030	Turkey	135	Zarghan	Iran

Table 2. Primers used for sequence-related amplified polymorphism (SRAP) analysis in safflower accessions.

Forward primers	Sequence (5^/ to 3^/)	Reverse primers	Sequence (5^/ to 3^/)
Me1	TGAGTCCAAACCGGATA	Em1	GACTGCGTACGAATTAAT
Me2	TGAGTCCAAACCGGAGC	Em2	GACTGCGTACGAATTTGC
Me3	TGAGTCCAAACCGGAAT	Em3	GACTGCGTACGAATTGAC
Me19	TGAGTCCAAACCGGTTG	Em4	GACTGCGTACGAATTTGA
Me21	TGAGTCCAAACCGGTCA	Em5	GACTGCGTACGAATTAAC

Table 3. SSR markers used for genetic diversity analysis in safflower accessions.

Primer	Repeat motif	Tm (^oC)	Primer sequence (5/-3/)	Reference
GB-CT-042	(AACCTC)4	58	CTCAACCCATCTCAGCCA CCTTCCCTTTGTCCTTCG	Lee et al. [25]
GB-CT-057	(AG)6	58	TCGGTCTGACGCTCTGAT GGAAGCAACTGCTTGTAGGA	Lee et al. [25]
GB-CT-081	(CTT)9	58	CAGACGCTGATGGGGTAG TTCCCCAGCTGTACCTCC	Lee et al. [25]
GB-CT-176	(TG)5	55	ATTGGCAAATGAACGGTG TGGCATACAAGGTGGGAA	Lee et al. [25]
GB-CT-184	(GAT)2,(GAT)3	55	GAGGAGGTCGAAAGCCTG TGGTAAAGGGTTGAGGGC	Lee et al. [25]
GB-CT-264	(GT)4AT(GT)4	55	AGGGAAGGTCTCAAAGGC AAACCCAGATCCTTTGGC	Lee et al. [25]
GB-CT-294	(GT)3(GCGTGT)2	55	GTTCGATAGTTTGGGGGC AAATCCCTGCCTCTCTGG	Lee et al. [25]
GB-CT-329	(ACAT)5	55	TGTCCTTACGGCTCTGTACC CAAAAAGGGCCCTCTGAC	Lee et al. [25]
GB-CT-400	(AAG)4	55	AAGCCAAAGAGAATCCATGA TTCCTCAACCTGCTTTGC	Lee et al. [25]
GB-CT-445	(ATGGC)4	55	GCCTAGAAGGACCAAGGC CATGCGAGGTTTTAAGCG	Lee et al. [25]

Figure 1. Cluster analysis of 135 safflower accessions based on morphological traits data across two years (2015-2016 and 2016-2017). The x–y coordinates are the principal components, which are linear combinations of the original dimensions.

Table 4. Descriptive statistics for agro-morphological traits in 135 safflower accessions evaluated in the field in 2015-2016 and 2016-2017.

	NSB	NCP	TKW	PH	RWC	DPH	NSC	OC	SY	NSP
Mean	7.90	11.93	37.94	78.73	43.35	21.28	25.09	29.54	11.30	287.29
Max	19.00	41.00	61.80	100.00	80.00	33.37	48.43	42.06	63.06	747.10
Min	3.80	5.00	23.80	57.00	18.06	15.50	8.67	20.02	2.64	78.02
Median	7.00	10.50	36.40	79.00	44.69	21.00	24.12	29.68	9.69	254.94
Variance	7.82	25.30	65.87	62.06	143.43	7.72	65.92	13.96	51.41	17909.99
CV (%)	12.72	14.63	16.78	17.23	19.75	12.62	19.64	14.23	18.78	10.12
LSD (5%)	1.14	3.83	2.49	7.98	2.98	1.94	3.11	1.25	3.29	18.89

NSB, number of secondary branches; NCP, number of capitula per plant, TKW, 1000-seed weight (g); PH, plant height (cm), RWC, relative water content; DPH, diameter head per plant (cm); NSC, number of seeds per capitula; OC, seed oil content (%); SY, seed yield (g/plant); NSP, number of seeds per plant.

Figure 2. PCR amplification profile obtained with SRAP marker (Em2-Me1) (a) and SSR marker (GBCT-184) (b) in safflower genotypes. M: molecular size marker (Cinagen, Iran).

Figure 3. Cluster analysis of 135 safflower genotypes based on genetic dissimilarity matrix obtained by SRAP markers.

Table 5. Polymorphism characteristics of 10 SRAP primer combinations in 135 safflower accessions.

Primer combination	Number of amplified bands	Number of polymorphic bands	Polymorphism (%)	Gene diversity	PIC	I
Em1-Me1	17	12	70	0.27	0.22	0.45
Em1-Me21	15	11	73	0.34	0.27	0.50
Em2-Me1	20	14	70	0.37	0.29	0.50
Em2-Me2	16	12	75	0.25	0.2	0.39
Em2-Me3	17	13	76	0.29	0.23	0.44
Em2-Me21	15	10	66	0.3	0.24	0.47
Em3-Me1	14	10	71	0.29	0.23	0.44
Em3-Me3	17	13	76	0.18	0.16	0.30
Em4-Me19	18	13	72	0.33	0.26	0.50
Em5-Me21	16	12	75	0.26	0.21	0.40
Mean	16.50	12	72.40	0.29	0.23	0.43

Figure 4. Cluster analysis of 135 safflower genotypes based on genetic dissimilarity obtained by SSR markers.

Table 6. Genetic parameters of the 10 polymorphic SSR markers used for genetic diversity in safflower.

Marker	Na	Ne	Major allele frequency	Nei’s index	I	Observed heterozygosity	Expected heterozygosity	PIC
GBCT-042	8	4.75	0.34	0.79	1.12	0.20	0.79	0.76
GBCT-057	5	4.38	0.32	0.77	1.54	0.22	0.77	0.73
GBCT-081	6	3.24	0.43	0.69	1.34	0.30	0.69	0.64
GBCT-176	8	3.42	0.42	0.70	1.44	0.29	0.70	0.66
GBCT-184	9	3.60	0.36	0.72	1.54	0.27	0.72	0.67
GBCT-264	5	3.56	0.44	0.71	1.43	0.28	0.72	0.68
GBCT-294	8	4.87	0.29	0.79	1.71	0.20	0.79	0.76
GBCT-329	4	2.24	0.61	0.55	1.02	0.44	0.55	0.50
GBCT-400	7	3.73	0.39	0.73	1.54	0.26	0.73	0.69
GBCT-445	8	5.25	0.27	0.80	1.79	0.19	0.81	0.78
Mean	6.8	3.90	0.39	0.73	1.51	0.26	0.73	0.69

Figure 5. Cluster analysis of 135 safflower genotypes based on genetic dissimilarity obtained by combined data (SRAP and SSR markers).

Figure 6. K values based on three independent runs and K ranging from 1 to 10 based on 135 safflower genotypes using SRAP and SSR markers.

Figure 7. Population genetic diversity of 135 safflower genotypes based on SRAP and SSR markers.

Figure 8. Bayesian information criterion (BIC)(a), the F statistic of the DAPC (b), Scatter plot from the DAPC (c) and the percentage of cumulative variance for the retained PCA eigen vectors (d) for 135 safflower accessions that were used to determine the optimal k number of clusters.

Lee GA, Sung JS, Lee SY, et al. Genetic assessment of safflower (Carthamus tinctorius L.) collection with microsatellite markers acquired via pyrosequencing method. Mol Ecol Resour. 2014;14(1):69–78.

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