Association of candidate genes for fatty acid content in soybean by temperature-switch PCR (TSP) genotyping

Bueno, Rafael Delmond; God, Pedro Ivo Vieira Good; Prata, Isadora Oliveira; Pereira, Pedro Henrique Scarpelli; Teixeira, Arlindo Inês; Piovesan, Newton Deniz; Barros, Everaldo Gonçalves de

doi:10.1590/1984-70332018v18n3a37

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ARTICLE • Crop Breed. Appl. Biotechnol. 18 (03) • Sept 2018 • https://doi.org/10.1590/1984-70332018v18n3a37 copy

Association of candidate genes for fatty acid content in soybean by temperature-switch PCR (TSP) genotyping

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

The development of molecular markers is essential for improvement of soybean cultivars with modified fatty acid content. The objective of this study was to identify and validate SNP markers in candidate genes for fatty acid content in soybean. Six candidate genes (ARAF, PDAT, ABI3, FAD2-1b, FAD3B, and FAD3C) were selected. Alignment of gene sequences identified 25 SNPs and 3 INDELs. TSP primers were used to identify SNP alleles. 259 recombinant inbred lines (RILs) (FA22 / CD219) and 185 F₂ progenies (A29 / Tucunaré) were tested for association of SNPs. An SNP for FAD3B was associated with variation in content of linoleic acid (R² = 5.84%) and linolenic acid (R² = 6.79%). In FAD3C, an SNP was associated with linoleic and linolenic acids (R² of 9.21% and 18.51%, respectively). The ABI3 gene was associated with palmitic acid, with R² = 5.41%. The SNP markers identified will be used in assisted selection for improvement of fatty acid content.

Key words:
Low linolenic acid; MAS-marker-assisted selection; SNP - single nucleotide polymorphism

Gene	Chromosome	Genic region	SNP alleles		LS^c Primer	AS Primer ^a,b	SNP allele tested
ABI3	18	5’UTR	A/C	Fwd	ATCCTATGAAGGGATGCAACA	CGGTATGATTATCATTGTGTATAT	A
				Rev	GCGTTTTGATCATCTTCGAGT
ARAF	3	5’UTR	A/T	Fwd	CCCCCTTCCTTTAACTCCTTATT	GGACTCTGATTCTGTTACTGTTA	A
				Rev	ACCCACCCACACCATGTTAC
FAD2-1	20	3’UTR	C/T	Fwd	AGGCAACCAATGCAATGAAG	GGTACAAGCTGTTTCACAATA	T
				Rev	TGGAGTGTGCATTTAAGGAAGAA
FAD3B	2	Exon	G/A	Fwd	GGATGAGTCATGGGTTCCAG	CCGTTTATGTGTCATTATCTTAG	G
				Rev	CCGGGGCTTCTTGAAAACTA
FAD3C	18	Exon	C/T	Fwd	GCAGTTTTGCCATCCTTCAA	ACAATTTGAGGGAAAAGGTA	T
				Rev	TTGACTTAGATCACATAGGCAGACA
PDAT	17	5’UTR	C /T	Fwd	GCTTGTCAGACATGAAACGTCC	CGATCAGATAGATCATTTAAGACT	T
				Rev	GGCAATTAAGCGGGAATGAA

Line/Population	Statistic	Fatty Acid
Line/Population	Statistic	Palmitic	Stearic	Oleic	Linoleic	Linolenic
FA22	mean	9.81	2.7	38.57	40.68	8.24
	sd	0.3	0.3	4.04	3.39	0.7
	min	9.24	2.09	30.97	35.59	7.11
	max	10.24	3.16	44.65	46.88	9.36
	cv (%)	3.06	11.11	10.47	8.33	8.50
CD219	mean	10.46	3.97	19.27	48.34	8.42
	sd	0.6	0.37	1.29	1.72	0.62
	min	10.67	3.66	18.91	51.06	8.5
	max	12.33	5.05	22.91	56.34	10.57
	cv (%)	5.74	9.32	6.69	3.56	7.36
A29	mean	11.01	4.07	21.72	62.03	1.19
	sd	0.47	0.452	3.22	3.41	0.175
	min	9.66	3.05	15.22	54.67	0.90
	max	11.88	5.11	28.33	69.85	1.85
	cv (%)	4.27	11.10	14.84	5.50	14.69
Tucunaré	mean	11.41	3.11	19.14	58.18	8.00
	sd	0.357	0.262	1	0.828	0.728
	min	10.91	2.71	17.08	56.57	6.00
	max	12.17	3.67	21.96	59.36	9.07
	cv (%)	3.13	8.43	6.23	1.42	9.11
FA22 x CD219	mean	8.80	2.70	35.73	46.87	5.90
	sd	0.85	0.66	4.06	2.78	1.02
	min	8.59	1.87	12.81	36.46	6.39
	max	15.16	5.87	46.38	60.75	12.35
	cv (%)	9.66	24.44	11.36	5.93	17.29
	D¹	0.03	0.05	0.065	0.067	0.04
A29 x Tucunaré	mean	10.33	3.67	24.74	57.04	4.21
	sd	1.45	1.28	4.37	4.02	1.59
	min	0.67	2.27	9.66	37.06	0.97
	max	14.23	19.97	47.89	65.92	12.9
	cv (%)	14.04	34.88	17.66	7.05	37.77
	D¹	0.05	0.05	0.06	0.045	0.067

Fatty Acid	Palmitic			Stearic			Oleic			Linoleic			Linolenic
Marker	F test p-value	Mean	R² _%	F test p-value	Mean	R² _%	F test p-value	Mean	R² _%	F test p-value	Mean	R² _%	F test p-value	Mean	R² _%
FA22 x CD 219
ARAF	1	8.78	0.15	1	2.69	0.0001	1	35.89	0.03	1	46.75	0.01	1	5.90	0.12
PDAT	1	8.78	0.006	1	2.69	0.1038	1	35.92	0.18	1	46.70	0.32	1	5.90	0.25
A29 x Tucunaré
ABI3	0.0055**	11.18	5.42	1	4.14	0.015	0.145	17.33	1.52	1	60.99	0.02	1	6.35	0.34
FAD3B	1	11.14	0.37	0.1905	3.90	1.38	0.1302	17.84	1.22	0.003**	61.80	5.84	0.002**	5.32	6.79
FAD3C	0.1505	11.16	1.13	1	3.99	0.01	1	17.54	0.0146	0**	61.17	9.21	0**	6.14	18.51

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Population	FA22 x CD219							A29 x Tucunaré
Population	Genotypic class^§						Genotypic class ^§
Marker	0	1	2	Hypothesis	χ2	P-value (%)	0	1	2	9	Hypothesis	χ2	P-value (%)
ARAF	126	2	131	1:1	0.10	75.5	-	-	-	-	-	-	-
PDAT	115	5	139	1:1	2.27	13.2	-	-	-	-	-	-	-
ABI3	-	-	-	-	-	-	38	66	38	44	1:2:1	0.70	70.32
FAD3B	-	-	-	-	-	-	38	100	48	-	1:2:1	2.13	34.49
FAD3C	-	-	-	-	-	-	39	90	54	-	1:2:1	2.508	28.53