Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty acid desaturase mutant alleles and its effect on seed germination along with other seedling traits.

Peanut (Arachis hypogaea L.) is an important nutrient-rich food legume and valued for its good quality cooking oil. The fatty acid content is the major determinant of the quality of the edible oil. The oils containing higher monounsaturated fatty acid are preferred for improved shelf life and potential health benefits. Therefore, a high oleic/linoleic fatty acid ratio is the target trait in an advanced breeding program. The two mutant alleles, ahFAD2A (on linkage group a09) and ahFAD2B (on linkage group b09) control fatty acid composition for higher oleic/linoleic ratio in peanut. In the present study, marker-assisted backcrossing was employed for the introgression of two FAD2 mutant alleles from SunOleic95R into the chromosome of ICGV06100, a high oil content peanut breeding line. In the marker-assisted backcrossing-introgression lines, a 97% increase in oleic acid, and a 92% reduction in linoleic acid content was observed in comparison to the recurrent parent. Besides, the oleic/linoleic ratio was increased to 25 with respect to the recurrent parent, which was only 1.2. The most significant outcome was the stable expression of oil-content, oleic acid, linoleic acid, and palmitic acid in the marker-assisted backcrossing-introgression lines over the locations. No significant difference was observed between high oleic and normal oleic in peanuts for seedling traits except germination percentage. In addition, marker-assisted backcrossing-introgression lines exhibited higher yield and resistance to foliar fungal diseases, i.e., late leaf spot and rust.

Introduction

Peanut or groundnut (Arachis hypogaea L.) is one of the world’s most important legumes for its valuable edible oil and protein content. It is a major cash crop and plays an essential role in the livelihood of millions, especially in semi-arid tropics. It is cultivated globally in around 27.94 million ha with a total production of 47.09 million tons [1]. China, India, Nigeria, and the United States of America are the leading groundnut producers that account for ~70% of the global peanut production. Peanut is traditionally used for the extraction of oil for edible as well as industrial purposes but the quality attributes vary with geographical region. In China, India, and other Asian countries, half of the produce is crushed for oil extraction and the rest is being used for confectionary and food purposes. While in the USA and other European countries more than two-thirds of peanut production are used for confectionary and food purposes and remaining one-third is used in the extraction of oil. Low oil content peanuts are preferred for table purposes and other food preparations of low caloric value.

Different proportions of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) determine the nutritional quality, shelf life, and flavor of peanut oil as well as its products. The peanut oil contains 80% unsaturated fatty acids (UFAs), mainly oleic (MUFA), and linoleic (PUFA) acids, whereas the remaining 20% SFAs comprises of palmitic, stearic, arachidic, behenic and lignoceric acid. Palmitic acid alone contributes half of the total SFAs while the rest five make up the remaining 50% [2]. SFAs are considered to increase serum low-density lipoproteins cholesterol level in the blood [3]. An elevated level of palmitic acid in the oil also increases the risk of cardiovascular diseases (CVD) [4]. A higher proportion of linoleic acid results in off flavors, rancidity, the short shelf life of oil and its derived products, which makes it undesirable for cooking purpose [5]. From a nutritional point of view, MUFA have been desirable in lowering plasma cholesterol levels and reduced risk of CVD [6, 7]. Therefore, a diet with high oleic (HO) acid can reduce the risk of heart diseases, inflammatory diseases tumorigenesis, and slow down atherosclerosis [8, 9]. In addition, oleic acid has ten-fold higher auto-oxidative stability than linoleic acid [10]. Therefore, there is a greater demand for the improved lines with higher oleic/linoleic (O/L) ratio in the peanut oil.

In peanut, fatty acid desaturase enzyme catalyzes desaturation of oleic to linoleic acid. [11, 12]. It is controlled by two homeologous genes ahFAD2A and ahFAD2B, located on A-genome (linkage group a09) and in B-genome (linkage group b09), respectively [13,14]. Mutations in ahFAD2A and ahFAD2B genes results in reduced fatty acid desaturase enzyme activity that leads to higher accumulation of oleic acid [13,15]. A single base pair (bp) substitution mutation (G:C to A:T) in ahFAD2A gene at 448 bp position results in a missense amino acid from aspartic acid to asparagine (D150N). While, an insertion mutation in A:T of ahFAD2B gene at 442 bp position generates premature stop codon [11, 12]. Thus the two mutant fatty acid desaturase alleles stop the conversion of oleic acid to linoleic acid in peanut [16, 17, 18]. Improved breeding lines with HO and lower linoleic and palmitic acids in peanut oil are essential to make peanut of superior quality. Norden et al., [19] first identified F435 as a natural peanut mutant line with approximately 80% oleic acid and 2% linoleic acid. Later on, the first ever HO peanut breeding line, SunOleic95R, was produced with the help of conventional breeding method in the USA [16]. Chen et al., [20] and Chu et al., [13] developed linked allele specific-polymerase chain reaction (AS-PCR) and cleaved amplified polymorphic sequence (CAPS) markers, respectively for both of the ahFAD2 alleles. The development of the associated markers in peanut helped in the improvement of ‘Tifguard High O/L’ variety in the USA through marker-assisted backcrossing (MABC) [21]. Recently, Janila et al., [22] introgressed ahFAD2 alleles from SunOleic95R into the elite breeding lines using MABC and marker-assisted selection (MAS) at ICRISAT, Patancheru, India. Further, Bera et al., [23] developed HO peanut lines through MAS at ICAR-Directorate of Groundnut Research, Junagadh, India. Most of these molecular breeding lines are under examination in All India Coordinated Research Project on Groundnut (AICRP-G) and, recently, Girnar 4 and Girnar 5 genotypes have been identified for release in India.

Peanut is grown in both rainy and post-rainy (as winter and summer crop) seasons across different states of India, varying largely in climatic and edaphic conditions. The chemical composition of peanut oil is influenced by several factors like genotype, geographic location, season, soil humidity, temperature and growing conditions [24, 25,26]. In general, lower temperature (22˗29°C) is associated with more linoleic acid synthesis due to increased activity of oleate desaturase enzyme [27, 28] and high temperature (30˗33°C) during pod filling to harvesting stage reduces the linoleic acid content in peanut oil [29, 30, 31]. Li et al., [32] also reported that season and temperature had a significant influence on fatty acid content in Brassica crops. Flagella et al., [33] reported a reduction in oleic and stearic acid while an increase in linoleic and palmitic acid in sunflower under irrigated cultivation. Furthermore, healthy and vigorous seedlings are one of the important criteria for making HO peanut cultivation profitable. The chemical composition of seed reserve might affect its germination and seedling vigor as seed reserve content is correlated with germination percentage [34]. In oilseeds, the major storage reservoir is lipid that provides essential energy to the growing embryo and thus affects seed germination. The alterations in seed lipid affect membrane lipid composition in respect to membrane function and permeability, which affects germination, vigor, and tolerance to environmental stress [35]. In peanut, germination percentage decreases with increase in O/L and unsaturated/saturated fatty acid ratios especially at lower (16°C and 14°C) temperatures [36]. Sun et al., [35] found that seed vigor of high oleate lines was lower as compared with the lines with normal oleic content in peanut. Upadhyaya et al., [37] reported a poor yield of ICG-2381, a groundnut accession with high O/L ratio.

Considering the demand of peanut with HO both in domestic and international markets, the present study was undertaken with three objectives: i) introgression of ahFAD2 alleles into the higher oil content peanut variety through MABC; ii) multi-location testing of MABC derived HO peanut lines over the two seasons for yield and impact of locations and seasons on the oil quality and oil content iii) determining the effect of HO trait on seed germination and other seedling traits.

Materials and methods

Plant material

For improving the oil quality, ICGV06100 was used as female/recurrent and SunOleic95R as male/donor parents for MABC breeding program. ICGV06100 is a high yielding and high oil containing (~55%) peanut line but with lower oleic acid (~39.3%), developed by ICRISAT, Patancheru, India (ICRISAT, 2012; unpublished). It is a Virginia bunch (semi-spreading) cultivar derived from the cross [(ICGV92069 × ICGV93184) × (NCAc-343 × ICGV86187) × S23]. SunOleic95R, having both ahFAD2 mutant homozygous alleles with HO (~80%) but lower yield and oil contents (~45%) was used as a male/donor parent. It was developed by Florida Experimental Agriculture Station, USA, from the mutant line F435 [16].

Under the second objective, MABC lines were tested for pod yield in multiple seasons and locations. Initial yield evaluation of MABC lines along with elite cultivars (Abhaya, CO-6, GG-20, ICGS-1043, GPBD-4, JL-24, TMV-2, VRI-6, K-6, TAG-24, GJG-31, and TG-37A) was done at a single location over two seasons. Subsequently, advanced yield evaluation of MABC lines together with other breeding lines and elite cultivars was done at three different locations. Besides fatty acids profile, oil and protein contents of MABC lines, SunOleic95R and ICGV06100 were also estimated at three locations.

For the accomplishment of the third objective, two separate panels of peanut genotypes were studied for seed and seedling traits. The first panel consisted of normal oleic acid peanut genotypes (GG-20, ICGV06100, ICGV05141, and ICGV06110), while the second panel had HO peanut genotypes (NRCGCS-587, HOP-IL_MAS-191, HOP-IL_MAS-145and HOP-IL_MAS-130) [22, 23, 38].

Molecular markers

Two types of DNA-markers linked to ahFAD2 mutant alleles were used for genotyping. The allele specific-polymerase chain reaction (AS-PCR) markers [20] were used to identify heterozygous plants for the mutant alleles. The cleaved amplified polymorphic sequence (CAPS) markers [13] were deployed to select homozygous plants for both the ahFAD2 alleles.

DNA extraction and marker genotyping

The DNA was extracted from tender fresh leaves of 10 to 15 days old field-grown seedlings using modified cetyltrimethylammonium bromide (CTAB) extraction method [39]. The quality and quantity of DNA were checked [25] and genotyping of the target population was done using AS-PCR and CAPS markers. The primer combination, F435-F and F435SUB-R, amplified 203bp fragment for the mutant allele (substitution from G:C→A:T, ahFAD2A) in the A-genome, while the primer combination, F435-F and F435INS-R amplified 195bp fragment for the mutant allele (A:T insertion, ahFAD2B) in the B-genome (Fig 1). In case of wild type ahFAD2A allele, the 826bp fragment was digested to 598bp and 228bp, while the mutant genotypes had the 826bp fragment intact. For B-genome, 2.0U of restriction enzyme Hpy188I (New England Biolabs, UK) was used for digestion of 10μl of PCR amplicon for about 16 hours at 37°C. The wild type ahFAD2B allele of 1214bp with five restriction sites cleaved into five fragments i.e., 736, 263, 171, 32 and 12bp.While the mutant allele had one additional restriction site in the 736bp fragment which was further cleaved into 550 and 213bp (all together six restriction sites in mutant instead of five in wild type) [23, 25].

Fig 1

AS-PCR assay, (a) Amplification of ahFAD2A mutant allele-specific 203 bp amplification in 1 to 5 F1 plants; (b) ahFAD2B mutant allele-specific 195 bp in3 to 4 while absent in 1 and 2 F1 plants; where SUN: SunOleic95R, M:100bp DNA ladder.

Estimation of background genome recovery and linkage drag

Eighty polymorphic single sequence repeats (SSRs) from 20 linkage groups (preferably two from each arm of a linkage group) were deployed to determine recurrent parent genome recovery in MABC lines [40, 41]. Furthermore, recurrent parent and MABC lines were assessed based on the passport data. Subsequently, the desirable recombinant plants possessing the smallest size of introgressed segments with minimum linkage drag among MABC lines were identified. For the analysis, additional 10 SSRs, selected from the ~20cM genomic region on either side of ahFAD2 loci from both a09 and b09 linkage groups, were used (S1 Table).

Hybridization and development of MABC lines

Hybridization was done at ICRISAT, Patancheru, India in 2011 during the rainy season. The crossed seeds were planted at ICAR-DGR, Junagadh in post-rainy season in the same year. F1s were genotyped with linked allele-specific markers to identify true F1 plants and plants heterozygous for ahFAD2 alleles were used for backcrossing. The BC1F1 plants were planted in 2012 rainy season and were genotyped with allele-specific markers to identify heterozygous plants at both the loci. Backcrossing and genotyping with AS-PCR markers were continued until the development of BC3F1 generation. The BC3F1 seeds were planted in 2013 rainy season and plants having ahFAD2 alleles were advanced to BC3F2 generation. The BC3F2 seeds were planted in 2013 post-rainy season and plants were genotyped with CAPS marker to identify plants with both the homozygous mutant loci. The BC3F2–3 plants homozygous for ahFAD2 alleles were advanced to BC3F3–4 in 2014 rainy season. Phenotyping for oil content and fatty acid composition was done in BC3F3–4progeny. Finally, introgression lines (ILs) were selected based on oleic acid content and was coded as MABC introgression lines (MABC-ILs).

Yield evaluation of MABC-ILs lines

The initial yield evaluation of MABC lines along with elite peanut cultivars was done in 2014 post rainy and 2015 rainy seasons. In both the seasons, genotypes were planted in a randomized block design (RBD) with three replications. The advanced yield evaluation of MABC-ILs along with other breeding lines and elite cultivars was carried out at three different states, namely Gujarat, Telangana, and Andhra Pradesh in both 2016 rainy and 2016 post-rainy seasons. The crops were sown in RBD with two replications. Each genotype was planted on four-meter beds in four lines. Recommended crop management practices were followed for raising a healthy crop. Pod yield per plot (7.2 m2) was recorded during the harvest on maturity of crop (111–115) days after sowing.

Biochemical analysis for oil content and fatty acid profile

The harvested mature kernels were subjected to oil and fatty acid analysis using Gas chromatograph (model number GC-700, Thermo Fisher, USA) [42] with flame ionization detector (FID) [23].

Seed and seedling traits

The matured kernels harvested from the plants of rainy season 2018 were subjected to the analysis. The pods harvested in the first week of October 2018 were sown in the third week of February 2019. The experiment followed RBD and was conducted in a BOD incubator (San-134, Sanco) under controlled temperature (32 ±2°C), humidity (70 ±5%), and cooled LED lights for 24 h. Each genotype was sown in five replications with 20 kernels per replication in randomized complete block design (RCBD). Ten kernels were sown in a UV protected 7×8 inch black-color plastic plant nursery bags, filed with normal soil (~2.3kg). Thus, two plastic plant nursery bags constituted single replication. The kernels were treated with Bavistin® (2 g per kg of kernels) prior to sowing. After sowing, watering was done until saturation of the polythene bags and kept in BOD for 15 days. Regular watering was maintained on every alternate day. Plastic bags were removed carefully after 15 days so that there was no damage to the root system. The individual plant was collected replication wise from each genotype after thorough washing (Fig 2). Observations on the rate of germination, shoot length, root length, shoot fresh weight, root fresh weight, plant dry weight, root dry weight, and vigor index were recorded. The rate of germination was calculated using the formula: Germination (%) = (number of seeds germinated/total number of seeds sown) × 100. Vigor index was calculated using the formula: Vigor Index = (Seedling dry weight× germination %) /100 [43].

Fig 2

Groundnut genotypes grown in BOD; a) Plants grown in polythene bags, b) Plants uprooted for recording observations on seedling traits.

Characterization of genotype

The passport data of MABC-ILs and recurrent parent were recorded on the basis of 16 qualitative and 17 quantitative traits, along with 6 special features, following peanut-descriptor [44] from five plant samples collected from the field at vegetative, reproductive, and harvesting stages.

Statistical analysis

Recurrent parent genome (RPG) recovery was analyzed using the formula: “RPG% = [{2 (R) + (H)}/2N] × 100” [45]; where “R” is the number of loci homozygous for recurrent parent allele; “H” is the number of loci still remaining heterozygous, and “N” is the total number of polymorphic markers used in the background analysis. The stability analysis for the pod yield was performed using AMMI ANOVA and GGE biplot models using R package [46]. A t-test was applied to assess the mean difference between oil, protein, moisture, oleic acid, linoleic acid, and palmitic acid contents among the MABC-ILs and parents. The significant differences between the mean values were determined by Duncan’s multiple range test (DMRT) (Duncan 1955) at a P ≤ 0.05 using CropStat version 7.2 [47]. Significant differences if any, between genotypes were compared using ANOVA.

Results

Development of advanced ILs through MABC

The crossed seeds received from ICRISAT, Patancheru, were planted at ICAR-DGR, Junagadh and resulted in 15 F1 plants. Eight plants were identified as true hybrids carrying both the mutant aFAD2 alleles. These eight F1 plants were used as pollen parents to make the first backcross with the recurrent parent. Out of 28 BC1F1 plants, six plants were found to carry both the ahFAD2 alleles in a heterozygous condition. Second backcrossing resulted in 32 BC2F1 plants and both the mutant alleles were found in nine plants. Third backcrossing resulted in 37 BC3F1 plants, among which six plants carried the ahFAD2 alleles. These six BC3F1 plants were selfed and 67 BC3F2 seeds were harvested and sown in the next season. BC3F2 plants were genotyped with the AS-PCR and CAPS markers, and three plants were finally identified as homozygous for both the ahFAD2 alleles (Fig 3). Subsequently, the fatty acid analysis confirmed single MABC-IL with ~80% oleic acid (which was later coded as NRCGCS-587).

Fig 3

CAPS assay; (a) Heterozygous and homozygous plants for ahFAD2A mutant allele; (b) Heterozygous and homozygous plants for ahFAD2B mutant allele; where M: 100bp DNA ladder, 1–6: MABC-ILs, P1: ICGV06100, P2: SunOleic95R, C: Control, ‘AA, BB’: homozygous wild alleles, ‘Aa, Bb’: heterozygous alleles and ‘aa, bb’: indicates homozygous mutant alleles.

Recurrent parent genome recovery and linkage drag

Eighty SSRs were polymorphic between the recurrent parents and NRCGCS-587. Homozygosity was found with 73 SSRs in NRCGCS-587 indicating 91.87% recurrent parent genome (RPG) recoveries. However, a genomic segment carrying the ahFAD2 alleles was present in NRCGCS-587. Out of the 10 polymorphic SSRs tested between SunOleic 95R and NRCGCS-587, nine SSRs were amplified only in NRCGCS-587 and not amplified in SunOleic 95R (S1 Table) indicating a linkage drag of additional segments away from the ahFAD2A and ahFAD2B alleles. Therefore, introgression of additional genomic regions in NRCGCS-587 resulted in some linkage drag but it showed no decrease in high oleic content.

Fatty acid profile analysis and estimation of oil content in MABC-IL (NRCGCS-587) and parents

Fatty acid profile analysis of NRCGCS-587 with its parents was done in two seasons (S2 Table). In 2014 post-rainy season plantations, oleic acid and linoleic acid contents in NRCGCS-587 were recorded as 78.8% and 4.0%, respectively. Whereas the same were 42.0% and 35.0% in the recurrent parent, respectively, and as 77.0% and 6.0% in the donor parent, respectively. The O/L ratio in NRCGCS-587 was 19.7, while it was 1.2 in the recurrent parent. The palmitic acid content was 6.8% in NRCGCS-587 as compared to 13.0% and 7.0% in the recurrent and donor parent, respectively (Fig 4). NRCGCS-587 contained 53% oil and 24% protein as compared to 54% oil and 26% protein in the recurrent parent and 48% oil and 26% protein in the donor parent (Fig 5). Further analysis of the oil content and fatty acid composition was done in 2015 rainy season. NRCGCS-587 showed 54% oil and 23% protein content, ICGV-06100 contained 54% oil and 24% protein, and SunOleic95R recorded 50% oil and 25% protein contents. So, there was no significant differences in oil and protein content of NRCGCS-587 with its parents. (Fig 5). In NRCGCS-587, oleic acid, linoleic acid, and palmitic acid contents were 81%, 3%, and 6%, respectively, as compared to 39%, 39%, and 9% in ICGV06100, and 80%, 3.0%, and 6.0%, in SunOleic95R, respectively (Fig 4). The O/L ratio was 27.0 in NRCGCS-587, while it was 1.0 in the recurrent parent and 23.25 in the donor parent.

Fig 4

Oleic acid, linoleic acid, and palmitic acid in NRCGCS-587 and parents grown in ICAR-DGR during 2014 post rainy and 2015 rainy; “*” indicates significance at 5%; “ns” indicates non-significant.

Fig 5

Oil, protein, and moisture in NRCGCS-587 and parents grown in ICAR-DGR during 2014 post rainy and 2015 rainy seasons; “*” indicates significance at 5%; “ns” indicates non-significant.

Pod yield of MABC-IL

NRCGCS-587, along with 12 elite cultivars, was tested for yield and related traits. The analysis of variance revealed significant differences among the genotypes and genotype × environment interaction for pod yield. In 2014 post-rainy season, pod yield of NRCGCS-587 was 1464 kg/ha that was significantly higher than the check cultivars Abhaya, CO-6, GG-20, ICGS-1043, JL-24, TMV-2 and VRI-6; on par with K-6, TAG-24 and GJG-31; and lower than TG-37A (Table 1). During 2015 rainy season, pod yield of NRCGCS-587 (1714 kg/ha) was significantly higher than check cultivars except for TG-37A and GG-20. The pooled pod yield of NRCGCS-587 (1589 kg/ha) was significantly higher than all the check cultivars except TG-37A. Shelling percentage (73%) and hundred-kernel weight (50g) of NRCGCS-587 were higher with the check cultivars. Besides, NRCGCS-587 was tested at three different states over two seasons. AMMI analysis of variance (Table 2) revealed a significant interaction effect of genotype × location on pod yield followed by location and genotype, individually. Stability analysis in all the three locations by GGE biplot showed that pod yield of NRCGCS-587 was higher (Fig 6) with local check cultivars in Telangana (ICGS76) and Andhra Pradesh (TCGS˗157) and superior to common check cultivar (GG˗20).

Table 1

Yield and the related traits of NRCGCS-587 grown in ICAR-DGR, Gujarat, during 2014 post rainy and 2015 rainy season.

Genotypes	Pod Yield (kg/ha)			Shelling (%)	100 kernel weight (g)
	2015 rainy	2014 post rainy	Mean
Abhaya	1418.4 c-d	1376.3 b-d	1397.3 b-d	72.1 a-c	49.3 b-d
Co-6	1485.4 c-d	1062.5 d-e	1274.0 b-e	70.7 a-d	57.3 a-b
NRCGCS-587	1714.0 b-c	1463.9 b-c	1588.9 b	72.1 a-c	59.0 a-b
GG-20	1883.0 a-b	967.9 e	1425.4 b-d	74 a-b	65.7 a
GJG-31	1488.2 c-d	1354.8 b-e	1421.5 b-d	66.4 d	51.3 b-d
GPBD-4	1485.0 c-d	1569.5 b	1527.2 b-c	74.1 a-b	52.7 b-d
ICGS-1043	1450.8 c-d	1178.5 c-e	1314.7 b-e	71.7 a-c	54.3 b-c
JL-24	1385.6 c-e	964.1 e	1174.9 c-e	69.7 b-d	46.0 c-e
K-6	1336.5 c-e	1499.4 b-c	1417.9 b-d	74.5 a	53.3 b-d
TAG-24	1271.6 d-e	1575.9 b	1423.8 b-d	70.9 a-d	49.3 b-d
TG-37A	2163.8 a	2105.5 a	2134.7 a	70.4 a-d	45.3 c-e
TMV-2	866.0 f	1122.4 c-e	994.2 e	72.8 a-c	44 d-e
VRI-6	1042.0 e-f	1038.1 d-e	1040.1 d-e	68.7 c-d	39.0 e
CV%	15.08	14.97	14.28	13.90	18.61

Means followed by same letter are not significantly different (less than or equal) at P = 0.05.

Table 2

AMMI Analysis of variance for pod yield evaluated at the three locations.

	df	MSS	Pr(>F)	% Sum of Squares
Locations (L)	5	4758501	<0.001	36.8
Rep (L)	6	125004	0.22	1.2
Genotype (G)	9	968802	<0.001	13.5
G*L	45	594607	<0.001	41.3
PC1	13	1174680	0	57.1
PC2	11	539693.9	0	22.2
PC3	9	349655.6	<0.001	11.8
PC4	7	241216.7	0.015	6.3
PC5	5	142883.3	0.165	2.7
Residuals	54	87212		7.3

PC1, PC2 …PC5 indicates principal components 1, 2….5 (denotes variation accounted by each components); df–Degrees of freedom; MSS- Mean sum of squares. P- value at 5%.

Fig 6

Average environment coordination (AEC) views of the GGE-biplot based on environment-focused scaling peanut genotypes evaluated for pod yield in Andhra Pradesh, Telangana, and Gujarat, India.

Oil content and fatty acid profile of MABC-IL in three different states

The pod samples of NRCGCS-587 were collected from three different states viz., Andhra Pradesh, Telangana and Gujarat in 2016 post-rainy season and subjected to biochemical analysis (S2 Table). Oil content in NRCGCS-587 did not differ much across the states, i.e., 54.7%, 54.5%, and 55.1% in Telangana, Andhra Pradesh, and Gujarat, respectively. Oleic acid content was almost the same in the pods of the two states, viz., Telangana (79.8%) and Andhra Pradesh (79.6%), while it was slightly higher in Gujarat (81.2%). Furthermore, linoleic acid (Telangana-3.0%, Andhra Pradesh-3.5%, and Gujarat-3.2%) and palmitic acid contents (Telangana-6.5%, Andhra Pradesh-6.4%, and Gujarat-7.8%) across the locations were similar (Figs 7 and 8). Likewise, oleic to the linoleic ratio in NRCGCS-587 also remained almost the same.

Fig 7

Oil, protein, and moisture in NRCGCS-587 and parents grown in Andhra Pradesh, Telangana, and Gujarat, India during 2016 rainy season.

Fig 8

Oleic acid, linoleic acid, and palmitic acid in NRCGCS-587 and parents grown in Andhra Pradesh, Telangana, and Gujarat, India during 2016 rainy season.

Passport data of NRCGCS-587 (MABC-IL) and recurrent parent

NRCGCS-587 is a Virginia bunch genotype characterized by decumbent-3 growth habit, alternate branching, green color, ovate leaf, and simple inflorescence. It takes about 23 days after germination for 50% flowering and 115 days for maturity. Average plant height, leaf length and leaf width are 42.6 cm, 40.1 mm, and 13.2 mm, respectively. It produces an average of five primary branches per plant and 2–3 flowers per inflorescence. Pods are mostly two seeded and the average length and width of pods are 26.0 mm and 12.4 mm, respectively. The mean length and width of kernels are 13.8 mm and 6.8 mm, respectively and it is rose in color (Fig 9). It yields 108.0 g of pods per square meter with 20% harvest index, 70% shelling-out-turn, ~55% oil content,~80% oleic acid, and ~4% linoleic acid content (S3 Table). Most importantly, NRCGCS-587 has also shown resistance to rust and late leaf spot, i.e., 1 and 3 disease severity scores, respectively in 1–9 modified scale (data not shown).

Fig 9

Plant, pod, and kernels of ICGV06100 and NRCGCS-587.

Average seed germination of 93.3% was found in normal oleic peanut, while it was 81.7% in HO peanut. A significant difference in germination percentage was recorded between normal and HO peanut (Table 3). There were no significant differences between normal and HO peanut for vigor index, fresh and dry plant weight, shoot and root length, fresh shoot and root weight, dry shoot and root weight, shoot length/root length, fresh shoot weight/fresh root weight, dry shot weight/dry root weight, and plant fresh weight/plant dry weight. However, the genotypic difference was observed within the normal and HO peanut groups. In both, the groups shoot length, fresh shoot biomass, and dry shoot biomass were higher than fresh root length, fresh root biomass, and dry root biomass.

Table 3

Details of seedling traits in normal oleic and high oleic peanut genotypes.

Trait	Name of genotypes	Oil%*	Oleic acid %*	Germination%	Shoot Length(SL)	Root Length(RL)	SL/RL	Fresh Shoot wt.(FSW)(g)	Fresh Root wt.(FRW)(g)	FSW/FRW	Dry Shoot wt.(DSW)(g)	Dry Root wt.(DRW)(g)	DSW/DRW	Plant Fresh wt.(PFW)(g)	Plant Dry wt.(PDW)(g)	PFW/PDW	Vigor index
High oleic(~80%) peanuts	NRCGCS-587	55	80	80.00	17.78	7.65	2.34	1.97	0.16	11.94	0.32	0.05	6.90	2.13	0.37	5.77	0.3
	HOP-IL_MAS-191	53.2	79.8	73.33	21.24	10.89	1.93	2.22	0.13	17.32	0.24	0.02	14.60	2.35	0.26	8.99	0.19
	HOP-IL_MAS-145	54.5	80.3	76.7	23.00	9.55	2.40	2.56	0.15	17.03	0.32	0.01	26.55	2.71	0.33	8.23	0.25
	HOP-IL_MAS-130	54.7	80.5	96.7	17.17	5.80	3.01	1.35	0.06	21.26	0.12	0.02	6.77	1.41	0.14	10.00	0.14
	Mean			81.70	19.79	8.47	2.42	2.02	0.13	15.98	0.25	0.02	13.71	2.15	0.28	7.81	0.22
Normal oleic (~50–55%)peanuts	GG-20	51	64	90	22.40	6.75	3.32	2.58	0.12	22.43	0.28	0.03	10.24	2.70	0.31	8.82	0.27
	ICGV-06100	55	39	83.30	15.46	5.93	2.63	2.02	0.35	5.77	0.27	0.05	6.36	2.37	0.32	7.38	0.26
	ICGV-05141	54.7	55	100.00	14.68	6.63	2.28	1.24	0.08	15.48	0.14	0.01	10.37	1.32	0.16	8.32	0.16
	ICGV-06110	53	38.3	100.00	17.20	4.15	4.42	1.62	0.06	29.36	0.10	0.01	20.40	1.67	0.11	15.61	0.11
	Mean			93.30	17.44	5.87	3.16	1.86	0.15	12.42	0.20	0.02	11.84	2.01	0.22	9.03	0.20
	CD@5%			7.55	5.16	1.42	0.9	0.73	0.04	3.65	0.1	0.01	5.51	0.76	0.1	2.57	0.08
	CV%			4.93	15.84	11.34	18.8	21.4	14.55	11.6	24.31	22.14	24.75	20.88	23.24	15.9	22.04

At 5% level of significance

*Source: [25, 26, 41]

Discussion

Peanut with HO is preferred over normal peanut due to its extended shelf life and multiple health benefits. High oil and oleic acid content in the peanuts are necessary for producing superior quality of oil to meet the nutritional needs and for industrial purposes. Moreover, the high oil containing peanuts can be used to combat malnutrition due to its higher caloric value over normal peanut. [48]. Therefore, improvement of oleic acid content in peanut for higher oxidative stability and better dietary properties is one of the important breeding objectives worldwide. Availability of molecular markers linked to the ahFAD2 gene has facilitated marker-assisted breeding for HO. MABC breeding further ensures the transfer of desirable gene together with maximum genome recovery of the recurrent parent [49, 50]. Previously, nematode resistance [51], rust resistance [52], and high oleic acid [22, 23] traits were transferred to elite peanut cultivars using MABC breeding. The use of CAPS and SNP markers has considerably reduced the time and volume of breeding material in different backcross generations [25]. In the first objective, a high oil content peanut genotype, ICGV06100, was targeted to improve oleic acid content using MABC breeding. The studies reported the development of a peanut genotype, NRCGCS-587, with high oil and HO content. The HO trait was introgressed from SunOleic95R into the genetic background of ICGV06100 through MABC approach and developed an improved version of ICGV06100 with 97% increase in oleic acid content over the recurrent parent.

The increase in oleic acid content in NRCGCS-587 led to a reduction in linoleic acid. There was a 90% and 24% reduction in linoleic acid and palmitic acid, respectively, in NRCGCS-587 as compared to the recurrent parent. Moreover, linoleic acid content ranged from 3.0% to 4.0% and palmitic acid ranged from 6.1% to 7.8% over different locations indicating their stable expression. The O/L ratio was increased to 27 in NRCGCS-587 from 1.2 in the recurrent parent. A similar trend of increase in oleic acid and O/L ratio, as well as a reduction in linoleic acid and palmitic acid, has already been reported [22, 23]. Commonly, an alteration in any of the metabolite biosynthesis also has a negative feedback effect on the production of other metabolites in a related pathway. Likewise, a significant reduction in palmitic acid level in NRCGCS-587 was recorded. Several previous studies have also reported a similar effect of ahFAD2 alleles on palmitic acid content [14, 22, 23, 53].

Generally, variation in oil content and fatty acid composition was reported in different environments due to the quantitative nature of these traits that are controlled by complex pathways [25, 26, 54]. However, limited or no variation was observed in NRCGCS-587 regarding oil, oleic, linoleic, and palmitic acid contents over locations indicating the minimal environmental effect on oil and HO traits. It seems that only a few independent genes, with the major effect, control oil and oleic acid production in NRCGCS-587. The selection for improved fatty acid composition would not affect the oil content of seed since there was no significant correlation between percent oil and any of the fatty acids or related variables [55]. Although fatty acid composition showed variation with the growth habit and environment, the oil content remained constant [56, 57, 58]. As a result, NRCGCS-587 with stable oil content across locations would be a better choice for use as a parent in the future breeding program on enhancing oleic acid and oil content in peanut.

NRCGCS-587 had more than 90% background genome recovery as well as precise introgression of ahFAD2 alleles. Moreover, identical passport data of NRCGCS-587 and ICGV06100 except oleic acid content corroborate maximum genome recovery from recurrent parent and precise introgression of ahFAD2 alleles in NRCGCS-587. Thus, NRCGCS-587 is an improved version of ICGV06100 having ~80% oleic acid content. The combined approach of both genotypic and phenotypic selections was found appropriate and effective in selecting improved lines [23, 59]. High oleic acid content did not affect seedling traits except the rate of germination. Significant variation in the rate of germination between HO and normal oleic peanut groups might be due to the alteration in lipid composition of seeds leading to changed membrane function and permeability. The germination decreased as O/L and unsaturated/saturated ratios increased in peanut, especially at lower (16°C and 14°C) temperatures [35]. Jungman and Schubert [36] reported that HO lines had lower seed vigor than their paired lines with normal oleic content. In general, the processes of germination initiates at a temperature below 15°C in peanut. Lower germination rate observed in HO peanut in this research might be due to the change in fatty acid composition since the temperature was maintained constant at 32°C. In sorghum, the α-amylase activity of seeds and subsequent seed germination percentage were affected by long-chain fatty acid composition [60].

In Pinus pinea, an increase in caprylic or oleic acids retarded the seed germination. The inhibition was dependent on fatty acid concentration and chain-length [61]. Short-chain fatty acids could infiltrate membrane lipids and change the physical properties that lower the seed germination [62].

In conclusion, there was a narrow but significant difference in seedling establishment between HO and normal oleic peanut under optimum temperature. Poor seed germination rate in HO peanut than normal peanut could be a cause of concern if a significant difference is more and needs further investigation to overcome it. A perfectly stable genotype having constant yield across geographical locations is a key to a successful variety [63]. The higher pod yield in the post-rainy season than a rainy season in NRCGCS-587 indicated that it might be more remunerative under irrigation than rain-fed conditions. It yielded either significantly higher or on par with all check cultivars except TG-37A indicating the potential to excel the local elite varieties from different peanut-growing states in India. Shelling percent and hundred-kernel weight were also on par with elite cultivars. Furthermore, NRCGCS-587 recorded maximum pod yield (2445 kg/ha) in Telangana and Andhra Pradesh that makes it suitable for these states. Stable pod yield, oil content, and HO content of NRCGCS-587 over the locations make it more rewarding for the peanut growing farmers. NRCGCS-587 is an improved version of ICGV06100 having genotypically 91% RPG and ahFAD2 alleles, and phenotypically high oil and yield. Thus, improved nutritional qualities would fetch premium price to the farmers without compromising the yield and meet the demand of peanut oil for industrial purposes.

Details of markers used in AS-PCR and CAPS analysis, background selection and testing of recombination in MABC line.

(XLSX)

Click here for additional data file.

Fatty acid profile MABC line and its parents.

(DOCX)

Click here for additional data file.

Qualitative, quantitative and special features of ICGV 06100 and NRCGCS-587 as per peanut descriptor.

(DOCX)

Click here for additional data file.

27 Aug 2019

PONE-D-19-15336

Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty acid desaturase mutant alleles and its effect on seed germination along with other seedling traits

PLOS ONE

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ACADEMIC EDITOR: You will see that both the reviewers made critical recommendations, but an important consideration is whether the study describes a technically sound research and made significant advances in the relevant area. However, as reviewers pointed out, I think that this manuscript needs additional works. Therefore, I encourage the authors to perform the revision as per the reviewer’s suggestions and if these were meticulously performed, then I am sure that the MS could be reconsidered on a later date.

==============================

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Reviewer #1: This manuscript was well written but it may not fit well to this journal. I gave this recommendation for three clear reasons: 1. The authors tried to describe a whole process for development of high oil and high oleate cultivars. The experiment was not specifically designed for any genetic studies or chemical analysis. 2. In the title, steady expression of high oleic acid and its effect on seed germination along with other seedling traits did not match the manuscript content. The evaluation of these traits was the selection outcome and did not necessary related to high oleic acid. 3. Even before making the cross, authors did not genotype parent ICGV06100 for FAD2 genotype. On Figure 2a and b, it was so confused. The well images were so different, but they scored them into the same genotype (for example, on Figure 2a well 6 and 7 to Aa; Figure 2b well 5, 6, and 7 images are the same but scored to different genotype Bb, BB, Bb). In addition, Figure 1a, only 4 samples but the authors mentioned 1 to 5. Figure 1b image was not clear. Furthermore, in the introduction (lines79-81), the authors mistook gadoleic acid (C20:1) as saturated fatty acid.

Reviewer #2: Results:

Table 1 results are not included in the results section

Line 274 to 282 is this data represented in one of the figures or tables?

line 291 what is the p-values for Figure 3?

Line 294 p-value?, Line 296 25% protein contents, should state "with no significant differences??"" and cite the p-value associated

Line 298 p-value

line 305 pod yield, p-value????

Line 311 shelling percentage in table 1 data, what are the stats and p-value??

Line 314 what are the p-values for the significant interaction effects??

Line 321 where is this data table???

Line 323 p-value is needed??

Line 328 p-value is needed to support statement

line 351 mention significant differences if any her in detail and in table 3, state p-value

Line 361 to 362 please re-word for clarity is confusing as written

Table 1, table footnotes are needed, define importance of superscripts statistically, what are the p-values

table 2 define in table footnote how yield mean sq was determined, describe in footnotes the 3 locations, define PC1, etc, briefly state in footnotes stats used

Table 3. table footnotes, table should in p-values, also, briefly state the methods used, stats used, so that reader can more clearly understand the data

Figure 1 and Figure 2 are never discussed in the results section. also if these figures are to be used they need to include figure legends with details of the methods and quantity of the DNA starting materials for amplification

Figure 3, Figure 4, Figure 6. and Figure 7, figure legend is needed, p-values needed with standard error bards, bar graphs using the current colors is difficult to read, try black and white and different patterns, % of what?? % of total fatty acid, x-axis values??

Figure 5???? not sure if this data adds to the strength of the manuscript??

Figure 8 same as previous comments of Figure 3, 4, also what are the harvest times and from what three states, figure legend needed

Figure 9. this figure is never discussed in the manuscript. either include and discuss in the results/discussion or remove. not sure if it adds to the strength of the manuscript.

General

Line 71 and "the" rest

Line 73 and the "remaining on-third"

Line 74 low caloric (corrected spelling)

line 105, remove "in a while"

line 295 space after and

line 297 space after and

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18 Sep 2019

Reviewer 1

Comment 1: The authors tried to describe a whole process for development of high oil and high oleate cultivars. The experiment was not specifically designed for any genetic studies or chemical analysis

Reply 1: We accept the comment partially.

Main aim of the experiment was to develop the introgression lines for high oleic traits which we have been developed successfully.

Once we developed the introgression lines then only we aimed for genetics studies and analysis of biochemical parameters and effect of high oleic acid on seed traits which are in sequence one after another. Hope This properly justify our experimentation.

Comment 2: In the title, steady expression of high oleic acid and its effect on seed germination along with other seedling traits did not match the manuscript content. The evaluation of these traits was the selection outcome and did not necessary related to high oleic acid.

Reply 2: We beg to differ from the comment of reviewer. The title of the manuscript clearly describe three components of studies conducted by us.

Component 1: Steady expression of high oleic acid in peanut

Component 2: (high oleic peanut) bred by marker-assisted backcrossing

Component 3: (High oleic acid ) its effect on seed germination along with other seedling traits

Hence, our manuscript exclusively describe the above three components and their results. Hence, the title clearly match the manuscript.

Comment 3:

Point-1: Even before making the cross, authors did not genotype parent ICGV 06100 for FAD2 genotype.

Point-2: On Figure 2a and b, it was so confused. The well images were so different, but they scored them into the same genotype (for example, on Figure 2a well 6 and 7 to Aa; Figure 2b well 5, 6, and 7 images are the same but scored to different genotype Bb, BB, Bb). In addition, Figure 1a, only 4 samples but the authors mentioned 1 to 5. Figure 1b image was not clear.

Point 3: Furthermore, in the introduction (lines 79-81), the authors mistook gadoleic acid (C20:1) as saturated fatty acid.

Reply 3: We beg to differ from the comment of reviewer.

Point-1: Genotyping of ICGV 06100 was very much included in the gel picture (Figure 2a and 2b). In the figure 2a and 2b lane marked as P1 depicts the recurrent parent-1 which is ICGV 06100. However, we regret to mention here that labelling in the figure 2a and 2b was not proper which we have corrected subsequently.

Point-2: We wholly accept the comments of reviewer for mismatching the labelling and quality of Fig. 1a, 1b 2a and 2b.

Fig (1a, 1b, 2a and 2b) have been replaced by clear gel pictures with proper and corrected labelling. Also scoring details of these gel pictures have been added in Materials and methods (DNA extraction and marker genotyping subheading) to make the scoring clear.

Point 3: Necessary correction has been made in the manuscript.

Reviewer 2

Comments 1: Table 1 results are not included in the results section

Reply 1: We accept the comment of reviewer. We unknowingly made mistake to mention table 1 in the manuscript which we have included under subheading “Recurrent parent genome recovery and linkage drag” of result section.

Comment 2: Line 274 to 282 is this data represented in one of the figures or tables?

Reply 2: We accept the comment of reviewer. We have made necessary correction and mentioned “Table 1” in the “Recurrent parent genome recovery and linkage drag” of result section

Comment 3: Line 291 what is the p-values for Figure 3?

Reply 3: We accept the comment of reviewer. Necessary correction has been included as P value at 5 % level of significance (added in footnote of Fig.3)

Comment 4: Line 294 p-value?, Line 296 25% protein contents, should state "with no significant differences??"" and cite the p-value associated

Reply 4: We accept the comment of reviewer. Necessary correction (one sentence of no significant difference as depicted in figure 4 has been added in manuscript). P value at 5 % level of significance (added in footnote of Fig.4)

Comment 5: Line 298 p-value

Reply 5: We accept the comment of reviewer. p-value is at 5 % level of significance (added in footnote in Fig.3)

Comment 6: line 305 pod yield, p-value????

Reply 6: We accept the comment of reviewer. p-value at 5 % level of significance added in the table 1.

Comment 7: Line 311 shelling percentage in table 1 data, what are the stats and p-value??

Reply 7: We accept the comment of reviewer. We have used Duncan’s Multiple Range Test (DMRT) for test of significance at 5%.

Comment 8: Line 314 what are the p-values for the significant interaction effects??

Reply 8: We accept the comment of reviewer. Significance at 5% included in table 2.

Comment 9: Line 321 where is this data table??? Line 323 p-value is needed?? Line 328 p-value is needed to support statement.

Reply 9: We accept the comment of reviewer. Results described in the results section is completely based on figure 6 and figure 7. We depicted the data in bar diagram only not mentioned in a fresh table. The numerical values of oil content, Oleic, Linoleic and palmitic acids between states were more or less similar. Hence neither we compared it statistically nor added as a separate table. Hence, no p-value is mentioned.

Comments 10: Line 351 mention significant differences if any her in detail and in table 3, state p-value

Reply 10: We accept the comment of reviewer. P-vale at 5% included in the Table 3.

Comment 11: Line 361 to 362 please re-word for clarity is confusing as written

Reply 11: We accept the comment of reviewer. We have edited the sentence to make to make meaning clear

Comment 12: Table 1, table footnotes are needed, define importance of superscripts statistically, what are the p-values

Reply 12: We accept the comment of reviewer. We have made necessary correction in the Table 1.

Comment 13: Table 2 define in table footnote how yield mean sq was determined, describe in footnotes the 3 locations, define PC1, etc, briefly state in footnotes stats used

Reply 13: We accept the comment of reviewer. We have made necessary correction in the Table 2. Name of three locations were mentioned in the write up, hence avoid in the table to avoid clumsiness.

Comment 14: Table 3. table footnotes, table should in p-values, also, briefly state the methods used, stats used, so that reader can more clearly understand the data

Reply 14: We accept the comment of reviewer. Footnotes added in the table 3. Methodology and stats used have been mentioned in the material methods section.

Comment 15: Figure 1 and Figure 2 are never discussed in the results section. also if these figures are to be used they need to include figure legends with details of the methods and quantity of the DNA starting materials for amplification

Reply 15: We accept the comment of reviewer. Results of Figure 2 was already discussed in the manuscript. However, results of Figure 1 was missing. We have added the results of Figure 1(with legends) in Materials and methods (DNA extraction and marker genotyping), methods of genotyping has also been added and references have been added.

Comment 16: Figure 3, Figure 4, Figure 6. and Figure 7, figure legend is needed, p-values needed with standard error bards, bar graphs using the current colors is difficult to read, try black and white and different patterns, % of what?? % of total fatty acid, x-axis values??

Reply 16: We accept the comment of reviewer. We have modified all the graphs accordingly. X- axis is self-explanatory (indicates % of total fat)

Comment 17: Figure 5???? not sure if this data adds to the strength of the manuscript??

Reply 17: We accept the comment of reviewer. Yes, Fig. 5 is needed as it indicates stability and high yield of NRCGCS-ILs over locations by GGE biplot analysis and to identify such genotype was ours second objective.

Comment 18: Figure 8 same as previous comments of Figure 3, 4, also what are the harvest times and from what three states, figure legend needed

Reply 18: We accept the comment of reviewer. Figure 8 is essential, as it shows the passport data of new identified line NRCGCS-587 (COMPARED WITH RECURRENT PARENT)

One sentence on harvest time i.e., Harvest on maturity of crop (110-115 days after sowing) has been added in Yield evaluation section of MABC-ILs in results section.

Comment 19: Figure 9. this figure is never discussed in the manuscript. either include and discuss in the results/discussion or remove. not sure if it adds to the strength of the manuscript.

Reply 19: We accept the comment of reviewer. Figure 9 has now been added in materials and methods part (seed and seedling traits), figure is a part of explanation of methodology followed to grow the seedlings to screen for important morphological traits to characterize the genotype.

Comment 20: General comments

Line 71 and "the" rest

Line 73 and the "remaining on-third"

Line 74 low caloric (corrected spelling)

line 105, remove "in a while"

line 295 space after and

line 297 space after and

Reply 20: We accept the comments of reviewer. We have incorporated all the corrections highlighted by the reviewer.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

5 Nov 2019

PONE-D-19-15336R1

Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty acid desaturase mutant alleles and its effect on seed germination along with other seedling traits

PLOS ONE

Dear Dr. Bera,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Thank you for submitting your revised manuscript. You will see that one of the reviewers make critical recommendations and it appears that additional work is needed as indicated by the reviewers. If these were meticulously performed, then I am sure that the MS could be reconsidered on a later date.

==============================

We would appreciate receiving your revised manuscript by Dec 20 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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Please include the following items when submitting your revised manuscript:

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Reviewers' comments:

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #3: The authors have responded to all questions raised by the previous reviewer except the question about the title. They need to change the title because it does not reflects the contents of manuscript.The title should mention the main achievement which is the development of high oleic content line. The title might read something like this "Development of stable lines with high oleic content through marker assisted introgression of fatty acid desaturate mutant alleles and its effect on seed germination". All other questions raised by previous reviewer has been answered and have improved the quality of manuscript significantly.

Reviewer #4: I feel that the title may be modified. Currently, the title seems to indicate that variations in germination frequencies and other seedling traits are primarily due to high oleic acid, which may not be the case due to various reasons including genomic reconstitution as elaborated below. It would be advisable to list these as separate features/studies of the high oleic lines.

In the abstract, the authors talk about resistance to biotic stresses but there is no mention of this data in the manuscript. This portion towards the end should be deleted.

Line 66: "in around"

I have the following four important queries which the authors need to clarify:

1. Lines 191-195: Aren't four SSRs per linkage group too less for estimating recurrent genome reconstitution?

2. Why was backcrossing done only till BC3? Would it be sufficient to reconstitute a significant portion of the recurrent parent genome in the absence of background selection.

Since background selection was not done, in conjunction to point 1 above, there could be sufficient portions of the non-recurrent parent genome remaining in the resulting selected line which could be responsible for other variations observed in seedling traits. Also, this would raise questions about the stability of the generated line.

3. Was stable inheritance of the high oleic phenotype tested using successive generations of harvested seed? This is not clearly given in the text. How was it done? Which generations of selfed progeny were used?

4. How is passport data a good approach to test for genomic reconstitution? I am not in agreement with this approach.

Line 226: Shouldn't it be "111-115"?

Line 260: Any references for this formula?

Line 284: Should be "codified" or "named" or "termed" instead of "decoded"

Line 290: (RPG) in brackets

Line 292: What does "nine were amplified" imply? Did they harbor the SunOleic95R genome segments? Is there any information available on the genetic/physical distance of these 10 SSRs?

Line 368-369: "However, the..........the groups" is not clear. Rephrase.

Figure 1 legend: Please correct for language

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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12 Nov 2019

Reviewer #3:

Comments:

The authors have responded to all questions raised by the previous reviewer except the question about the title. They need to change the title because it does not reflects the contents of manuscript. The title should mention the main achievement which is the development of high oleic content line. The title might read something like this "Development of stable lines with high oleic content through marker assisted introgression of fatty acid desaturate mutant alleles and its effect on seed germination". All other questions raised by previous reviewer has been answered and have improved the quality of manuscript significantly.

Reply:

We beg to differ from the comment made by the reviewer #3. The reviewer #3 mentioned that the authors had not responded to the question about the change of title which is absolutely incorrect. During the first review reviewer #1 offered the same comment in his comment no.-2. In reply we submitted justification for not making any changes in the title of our manuscript (Can be referred earlier review). We still stand with our justification this time also and do not see any point for making changes in the current title of our manuscript. However, final decision lies with the editor.

Reviewer #4:

Comment 1:

I feel that the title may be modified. Currently, the title seems to indicate that variations in germination frequencies and other seedling traits are primarily due to high oleic acid, which may not be the case due to various reasons including genomic reconstitution as elaborated below. It would be advisable to list these as separate features/studies of the high oleic lines.

Reply:

We beg to differ from the comment of the reviewer. The title of the manuscript clearly describe three components of the studies conducted by us.Component 1: Steady expression of high oleic acid in peanut; Component 2: Breeding of high oleic peanut by marker-assisted backcrossing;

Component 3: Effect of high oleic acid on seed germination along with other seedling traits. Hence, our manuscript clearly describe the above three components and their results. Hence, the title clearly match the manuscript.

Comment 2:

In the abstract, the authors talk about resistance to biotic stresses but there is no mention of this data in the manuscript. This portion towards the end should be deleted

Reply:

The data on resistance to biotic stresses (Rust and Late Leaf Spot diseases) of NRCGCS-587 and ICGV 06100 (recurrent parent) have been mentioned in supplementary Table–2 (S2). It may be treated as additional information. Hence, deleting the statement on resistance to biotic stresses from the text may not be required.

Comment 3:

Line 66: "in around"

Reply:

Accepted, Necessary correction has been made in the manuscript.

Comment 4:

Lines 191-195: Aren't four SSRs per linkage group too less for estimating recurrent genome reconstitution?

Reply:

It is always better to use SSRs as many as possible for estimating recurrent genome reconstitution. However, we selected four anchored SSRs from each linkage group (altogether 80 SSRs) which are genetically mapped in the peanut consensus map. Out of which 79 SSRs gave similar expression both in recurrent parent and introgression line. It gives a fair indication about the reconstitution of the recurrent parent genome in introgression line. Furthermore, we used three cycles of backcrossing which essentially (mathematically) recovers 93.75% of the recurrent parent genome. Both these approaches are supplementary to each of above two approaches and confirm recurrent parent genome reconstitution.

Comment 5:

Why was backcrossing done only till BC3? Would it be sufficient to reconstitute a significant portion of the recurrent parent genome in the absence of background selection. Since background selection was not done, in conjunction to point 1 above, there could be sufficient portions of the non-recurrent parent genome remaining in the resulting selected line which could be responsible for other variations observed in seedling traits. Also, this would raise questions about the stability of the generated line.

Reply:

Theoretically, three cycle of backcrossing supposed to recover 93.75% recurrent parent genome. We attempted three cycles of backcrossing which helped us to recover 93.75% recurrent parent genome. In such case taking background selection additionally would not yield any more information. However, one can do any number of additional work to strengthen the result. Yes, there is a chance of having 6.25% of non-recurrent parent genome in the introgression line and literally, this 6.25% of non-recurrent parent genome may throw little or no variations among the introgression lines.

Comment 6:

3. Was stable inheritance of the high oleic phenotype tested using successive generations of harvested seed? This is not clearly given in the text. How was it done? Which generations of selfed progeny were used?

Reply:

Yes, we have tested the high oleic phenotype in F3, F4 and F5 generations and confirmed.

It is clearly mentioned in the text line no. 300 (for the year 2014), 308 (for the year 2015) and 337 (for the year 2016 in multiplication).

High oleic trait was phenotyped through fatty acid analysis of kernels in Gas chromatography (Details methodology have been described in Materials and Methods section).

Selfed progenies of F3, F4 and F5 generations.

comment 7:

How is passport data a good approach to test for genomic reconstitution? I am not in agreement with this approach.

Reply:

Passport data of a genotype/verities of a particular crop is an important information which is used in DUS (Distinctness, Uniformity and Stability) testing. It helps to distinguish/identify one genotype from others. If two genotypes are similar in majority of the passport traits except one or two major trait(s), the genotypes can also be refereed as near isogenic line (NIL). Here, recurrent parent and introgression line are similar in majority of their passport traits except the high oleic content which we have introgressed into the introgression line. Theoretically, 3 cycle of backcrossing supposed to recover 93.75% genome of recurrent parent. Thus introgression line, NRCGCS-587 has got more than 90% genome constitution of recurrent parent (ICGV 06100), which has been reflected in the passport data of introgression line and recurrent parent.

Comment 8:

Line 226: Shouldn't it be "111-115"?

Reply:

Accepted, Necessary correction has been made in the manuscript.

Comment 9:

Line 260: Any references for this formula?

Reply:

Accepted, Necessary correction has been made in the manuscript.

Comment 10:

Line 284: Should be "codified" or "named" or "termed" instead of "decoded"

Reply:

Accepted, Necessary correction has been made in the manuscript.

Comment 11:

Line 290: (RPG) in brackets

Reply:Accepted, Necessary correction has been made in the manuscript.

comment 12:

Line 292: What does "nine were amplified" imply? Did they harbor the SunOleic95R genome segments? Is there any information available on the genetic/physical distance of these 10 SSRs?

Reply:

Accepted, Necessary correction has been made in the manuscript.

Yes, there is always possibilities, possible explanation is mentioned in the text.

Yes, Necessary information is already available in the supplementary table 1 (S1).

Comment 13:

Line 368-369: "However, the..........the groups" is not clear. Rephrase.

Reply:

Accepted, Necessary correction has been made in the manuscript.

Comment 14:

Figure 1 legend: Please correct for language

Reply:

Accepted, Necessary correction has been made in the manuscript.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

25 Nov 2019

Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty acid desaturase mutant alleles and its effect on seed germination along with other seedling traits

PONE-D-19-15336R2

Dear Dr. Bera,

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Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

Reviewer #4: (No Response)

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Reviewer #3: Yes

Reviewer #4: (No Response)

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Reviewer #3: Yes

Reviewer #4: (No Response)

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Reviewer #4: (No Response)

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Reviewer #3: I am surprised at stubborn attitude of authors. Three different reviewers have pointed out discrepancy in manuscript content and the title. The reasons have been cited and alternate titles have been suggested. Reviewers have been constructive in reviewing the manuscript but authors are reiterating the same comment again and again. They have not even considered what reviewers have been pointing out. Under these circumstances, I have no other comments to make and leave it to wisdom of editor to decide what will be right. I will not like to take up this manuscript for reviewing anymore.

Reviewer #4: (No Response)

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Reviewer #3: No

Reviewer #4: No

5 Dec 2019

PONE-D-19-15336R2

Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty acid desaturase mutant alleles and its effect on seed germination along with other seedling traits

Dear Dr. Bera:

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