Genetic variation of tocopherol content in a germplasm collection of Brassica napus L.

The genetic variation for tocopherol contents was investigated in a very divergent collection of 87 winter rapeseed genotypes grown in the greenhouse and in two years in the field. Genotypic and environmental effects were highly significant for alpha-, gamma- and total tocopherol contents and the alpha-/gamma-tocopherol ratio. Field and greenhouse environments differed significantly (p < 0.001) for tocopherol traits, with greenhouse means up to 19% higher than field means. Alpha-tocopherol content ranged from 63 to157 mg kg^-1 seed, gamma-tocopherol content from 114 to 211 mg kg^-1 seed, total tocopherol content from 182 to367 mg kg^-1 seed, and the alpha-/gamma-tocopherol contents ratio from0.36 to 1.23. The resynthesized lines often have a remarkably low alpha-tocopherol content and consequently a low alpha-/gamma-tocopherol contents ratio. The lines with altered fatty acid composition displayed the highest variation for both tocopherol content and composition. No significant differences were observed among groups with different seed quality types [00], [0+] and [++]. Total tocopherol content was not correlated with the alpha-/gamma-tocopherol ratio, indicating that total tocopherol content is independent from tocopherol composition. Alpha- and gamma-tocopherol contents were also not correlated. Gamma-tocopherol contents showed a significant positive correlation with oil content (r = 0.34^**). This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of tocopherol content and composition in winter rapeseed

The improvement of the nutritional value and the stability properties of rapeseed oil is partly hindered by the lack of information on the genetic control of tocopherols. The objectives of this investigation were to characterize the inheritance of tocopherols by using two factorial mating designs (North Carolina Design II, NC_II). The first (NC_II‐A) was produced with two sets of nine parents each, one selected for high and the other for low total tocopherol contents. In the second (NC_II‐B), each set consisted of six parents, which were either high or low for the α/γ‐tocopherol ratio. Parents and F₁ hybrids from both experiments were tested under field conditions in 1998 and 1999 using a completely randomized design with two replications. Only small differences were detected between mean values of parents and F₁ hybrids. General combining ability (GCA) variation in both experiments was highly significant (P < 0.01) for all tocopherol traits. Significant specific combining ability (SCA) effects were only detected for a‐tocopherol in NC_IIA and therefore for the α‐/γ‐tocopherol ratio. These results indicate that tocopherol contents and composition are controlled mainly by genes with additive effects. Interactions of F₁ hybrids and GCA effects with the environment were significant for only tocopherol contents and not for tocopherol composition.     

Mapping quantitative trait loci (QTLs) underlying seed vitamin E content in soybean with main,epistatic and QTL × environment effects

Soybean (Glycine max (L.) Merr.) seed contains small amounts of tocopherol, a non‐enzymatic antioxidant known as lipid‐soluble vitamin E (VE). Dietary VE contributes to a decreased risk of chronic diseases in humans and has several beneficial effects on resistance to stress in plants, and increasing VE content is an important breeding goal for increasing the nutritional value of soybean. In this study, quantitative trait loci (QTLs) underlying VE content with main, epistatic and QTL × environment effects were identified in a population of F_5 : 6 recombinant inbred lines from a cross between ‘Hefeng 25’ (a low‐VE cultivar) and ‘OAC Bayfield’ (a high‐VE cultivar). A total of 18 QTLs were detected that showed additive main effects (a) and/or additive × environment interaction effects (ae) in different environments. Moreover, 19 epistatic pairs of QTLs were found to be associated with α‐tocopherol (α‐Toc), γ‐tocopherol (γ‐Toc), δ‐tocopherol (δ‐Toc) and total VE (TE) contents. The QTLs identified in multienvironments could provide more information about QTL by environment interactions and could be useful for the marker‐assistant selection of soybean cultivars with high seed VE contents.     

Identification of quantitative trait loci and candidate genes controlling the tocopherol synthesis pathway in soybean (Glycine max)

A recombinant inbred line (RIL) population was used to identify quantitative trait loci (QTLs) and their candidate genes controlling the tocopherol (Toc) synthesis pathway. The RIL population was cultivated in field conditions in 3 years. A genetic map constructed using 1624 DNA markers was used for QTL analysis. We identified 22 QTLs for seed tocopherol contents and their ratios, of which two QTL clusters on chromosomes (Chr) 9 and 14 exerted consistent large effects on tocopherol composition across the 3 years. The QTL cluster localized on Chr 9 might correspond to γ-TMT3, which controls the conversion of γ-Toc into α-Toc. The QTL cluster localized on Chr 14 was novel, which might regulate the conversion of MPBQ (a precursor of δ-Toc) into DMPBQ (the precursor of γ-Toc). The effect of the QTL cluster on Chr 14 was validated in a pair of near isogenic lines, and its candidate gene was mined. The identified QTLs and their candidate genes might be used in breeding programmes to improve α-Toc content in soybean seeds.     

Identification of genetic variation in Brassica napus seeds for tocopherol content and composition using near‐infrared spectroscopy technique

Tocopherol is an essential fat‐soluble nutrient for humans. Increasing the tocopherol content in Brassica napus seeds can add value to rapeseed vegetable oil; this has become an important breeding target. However, there is no efficient and non‐destructive method for selecting rapeseed accessions with high tocopherol contents. Here, we report the first near‐infrared reflectance spectroscopy (NIRS)‐based technique for predicting rapeseed tocopherol content. Individual seed tocopherol compositions were estimated from 373 rapeseed genotypes of different origins. This method and chemical methods produced comparable predicted values of the tocopherol constituents in the seeds. Three equations were generated for the prediction of tocopherol content by using a modified partial least squares (MPLS) model. The total tocopherol content for the determination coefficient of cross‐validation (R²_cv) (0.74), determination coefficient (RSQ) (0.76) and one minus the ratio of unexplained variance to total variance (1‐VR) (0.65) values indicates a strong correlation between the calibration and validation sets. Overall, our model confirmed the NIRS method as feasible for predicting tocopherol content in rapeseed and as an efficient screening tool for future breeding programs.     

Genetic variability of tocopherol composition in sunflower seeds as a basis of breeding for improved oil quality

The variability of seed tocopherol content in wild sunflower species, the expressivity of tph1 and tph2 mutations in different lines and the oxidative stability of sunflower oil with altered tocopherol and fatty acid composition were objectives of this research. Near-isogenic lines for three genes, i.e. Tph1, Tph2, and Ol, were developed and investigated. Tocopherol content was determined with TLC and HPLC, as well as fatty acid composition with GC of methyl esters. Rancimat tests were used to estimate the oxidative stability of the oil. The seed tocopherol composition of wild sunflower species was shown to be uniform with a prevailing content of the α-homologue (90-99%). The genetic background of different near-isogenic lines was found to influence expressivity of mutations for tocopherol composition. High content of strong antioxidants, such as β-, γ-, and δ-tocopherols increased oil oxidative stability of linoleic and oleic types of oil by 1.2–3.0 times. The breeding model of sunflower hybrids should include antioxidant and vitamin parameters balanced for oils of different applications.     

Novel variation for the tocopherol profile in a sunflower created by mutagenesis and recombination

α‐Tocopherol is the main tocopherol in sunflower seeds (>90%). Because it exerts a weak antioxidant action in vitro, its partial replacement by other tocopherols is an important breeding objective in this crop. The objective of this research was to develop novel tocopherol profiles in sunflower through mutagenesis and genetic recombination. Seeds of four ‘Peredovik’ accessions were used for chemical mutagenesis with ethyl methane sulfonate (EMS). Single‐seed screening in the M₂ generation resulted in two M₂ seeds, derived from different M₁ plants, with increased γ‐tocopherol contents of 19.2% and 96.7%, respectively. M₃ progeny from the M₂ seed with the 96.7% content bred true for high c‐tocopherol content, containing more than 90%γ‐tocopherol. M₃ progeny from the M₂ seed with only 19.2%γ‐tocopherol segregated in a range from 0 to 84.6%. Selection for high c‐tocopherol content produced an M_4:5 line, designated IAST‐1, with a stable high concentration of γ‐tocopherol. Crosses between IAST‐1 and T589, with an increased b‐tocopherol content, produced F₂ segregants with trans‐gressive levels of up to 77%β‐tocopherol or up to 68% d‐tocopherol. Both novel tocopherol profiles were confirmed in the F₃ generation.     

Mapping of QTL controlling tocopherol content in winter oilseed rape

Tocopherols are natural antioxidants in vegetable oils and are important dietary nutrients. Enhanced tocopherol content has become an important objective in oilseed rape breeding. A segregating DH population was tested for 2 years at two locations in replicated field trials. Genotypic differences occurred for α‐, γ‐ and total tocopherol content as well as α/γ‐tocopherol ratio, but highly significant genotype x environment interactions resulted in low heritabilities. Using a mixed‐model composite interval mapping approach between one and five QTL with additive and/or additive x environment interaction effects could be mapped for α‐, γ‐ and total tocopherol content and α/β‐tocopherol ratio. In addition, one to six locus pairs with epistatic interaction effects were identified, indicating a strong contribution of epistasis to trait variation. In total, the additive and epistatic effects explained between 28% (α‐tocopherol content) and 73% (total tocopherol content) of the genotypic variance in the population, with individual QTL and locus pairs contributing between 7.5 and 29.2% of variance. Considering the low heritabilities of the tocopherol traits, the results of this study indicate that marker‐assisted selection may be an efficient strategy in a breeding program for enhanced tocopherol content in rapeseed.     

Variations in chlorosis and potential usefulness of alloplasmic Brassica rapa with the cytoplasm of male sterile Brassica juncea

To select superior seed parents for vegetable hybrid seed production, we conducted interspecific crosses between male sterile Brassica juncea (2n = 36, AABB) and eight inbred lines of Brassica rapa (2n = 20, AA). Alloplasmic lines of B. rapa with the cytoplasm of B. juncea were developed from B. juncea × B. rapa hybrids by repeated backcrossing using B. rapa as the recurrent male parent until the BC₃ generation. Seed fertility, male sterility and chlorophyll content were investigated in these plants cultivated under four different temperature conditions (5, 10, 12 and 20°C). At 10°C, the alloplasmic lines of B. rapa with the cytoplasm of B. juncea were male sterile with partly chlorotic leaves. The alloplasmic B. rapa had lower chlorophyll a, chlorophyll b and carotenoid contents than those of the original B. rapa. The leaves recovered from chlorosis when the plants were cultivated at 20°C. An alloplasmic line of B. rapa (A6) is available as a seed parent for vegetable hybrid seed production and contributes seed fertility, slight chlorosis and stable male sterility.     

Production of a desirable Brassica oleracea CMS line using an alloplasmic B. rapa CMS line carrying Diplotaxis erucoides cytoplasm as a bridge plant

In Brassica oleracea, production of F₁ hybrid seeds mainly makes use of the improved Ogura cytoplasmic male sterile (CMS) line. However, reliance on one particular line is a risk, and it would be advantageous to develop other CMS lines. In this study, we transferred Diplotaxis erucoides cytoplasm to B. oleracea cultivars using an alloplasmic B. rapaCMS line as a bridge plant to avoid incompatibility between donor and recipient plants. The new B. oleraceaCMS lines, which were derived by four generations of backcrossing, had small rudimentary anthers with no pollen grain and showed complete male sterility. There was no functional defect in other floral organs, and the ability to receive normal pollen did not appear to be impaired. Moreover, the B. oleraceaCMS lines carrying D. erucoides cytoplasm had larger leaf areas and a normal plastochron. As a consequence, the B. oleraceaCMS lines carrying D. erucoides cytoplasm have the potential to be valuable alternatives for use in commercial B. oleracea hybrid seed production.     

Breeding for the improvement of the ideal plant type of sesame

The objective of this study was to improve the ideal type of sesame, by performing pedigree selection applied to segregating generations of crosses of genotypes with contrasting characters. In the segregating F₂ population, the progeny were classified into eight types according to the combinations of carpel number per capsule, capsule number per axil and branching habit. Progeny of individual F₂ plants including a particular type were advanced to the F₆ generation. It was possible to improve the lines with eight types at the end of the selection process. Some types, especially bicarpels, monocapsule, branch (BMB) and bicarpels, tricapsules, branch (BTB), were considered as ideal plant types in breeding for high‐yielding varieties. While the low‐yielding type quadricarpels, tricapsules, non‐branch (QTN) was the highest in oil content (49.3%), the high‐yielding type BMB was the lowest (43.2%), Although the QTN‐type had the lowest content of oleic acid (41.3%) and the highest content of linoleic acid (43.1 %), the bicarpels, monocapsule, non‐branch (BMN) type had the highest content of oleic acid (48.4%) and the lowest content of linoleic acid (36.6%), Total tocopherol varied between 175.6 and 368.9 mg/kg in the seed oil of the sesame types. The best high‐yielding type BMB was one of the types containing less tocopherol.     

Molecular mapping of soybean seed tocopherols in the cross ‘OAC Bayfield’ × ‘OAC Shire’

Soybean (Glycine max [L.] Merr.) is cultivated primarily for its protein and oil in the seed. In addition, soybean seeds contain nutraceutical compounds such as tocopherols (vitamin E), which are powerful antioxidants with health benefits. The objective of this study was to identify molecular markers linked to quantitative trait loci (QTL) that affect accumulation of soybean seed tocopherols. A recombinant inbred line (RIL) population derived from the cross ‘OAC Bayfield’ × ‘OAC Shire’ was grown in three locations over 2 years. A total of 151 SSR markers were polymorphic of which a one‐way analysis of variance identified 42 markers whereas composite interval mapping identified 26 markers linked to tocopherol QTL across 17 chromosomes. Individual QTL explained from 7% to 42% of the total phenotypic variation. Significant two‐locus epistatic interactions were identified for a total of 122 combinations in 2009 and 152 in 2010. The multiple‐locus models explained 18.4–72.2% of the total phenotypic variation. The reported QTL may be used in marker‐assisted selection (MAS) to develop high tocopherol soybean cultivars.     

Mapping of male sterility gene ms10 in chilli pepper (Capsicum annuum L.)

Most of the hybrid seed in chilli are produced manually, but the use of male sterility (MS) can reduce the cost of hybrid seed production. MS‐12, a nuclear male‐sterile (NMS) line developed at Punjab Agricultural University, Ludhiana (India), has been utilized to develop commercial F₁ hybrids. A recessive gene, designated as ms10, governs MS in MS‐12. Due to recessive gene control, development of new NMS lines incorporating ms10 gene is tedious and time‐consuming. We identified SSR markers AVRDC‐PP12 and AVRDC_MD997* linked to the ms10 gene. A total of 558 primer pairs were screened following bulked segregant analysis (BSA). Linkage analysis in 210 F₂ plants indicated that the two SSR markers were linked to the ms10 gene and the marker AVRDC‐PP12 was closest to the gene at 7.2 cM distance. The marker was mapped to chromosome 1 at genome position 175 694 513 to 175 694 644. Until more closely linked markers are developed, the marker AVRDC‐PP12 would facilitate transfer of ms10 gene through marker‐assisted selection (MAS). Fine mapping would lead to cloning of the ms10 gene.     

Identification and validation of an over‐dominant QTL controlling soybean seed weight using populations derived from Glycine max × Glycine soja

Heterosis, or hybrid vigour, has been used to improve seed yield in several important crops for decades and it has potential applications in soybean. The discovery of over‐dominant quantitative trait loci (QTL) underlying yield‐related traits, such as seed weight, will facilitate hybrid soybean breeding via marker‐assisted selection. In this study, F₂ and F_2 : 3 populations derived from the crosses of ‘Jidou 12’ (Glycine max) × ‘ZYD2738’ (Glycine soja) and ‘Jidou 9’ (G. max) × ‘ZYD2738’ were used to identify over‐dominant QTL associated with seed weight. A total of seven QTL were identified. Among them, qSWT_13_1, mapped on chromosome 13 and linked with Satt114, showed an over‐dominant effect in two populations for two successive generations. This over‐dominant effect was further examined by six subpopulations derived from ‘Jidou12’ × ‘ZYD2738’. The seed weight for heterozygous individuals was 1.1‐ to 1.6‐fold higher than that of homozygous individuals among the six validation populations examined in different locations and years. Therefore, qSWT_13_1 may be a useful locus to improve the yield of hybrid soybean and to understand the molecular mechanism of heterosis in soybean.     

A Robertsonian translocation from Thinopyrum bessarabicum into bread wheat confers high iron and zinc contents

Development of wheat–alien translocation lines has facilitated practical utilization of alien species in wheat improvement. The production of a compensating Triticum aestivum–Thinopyrum bessarabicum whole‐arm Robertsonian translocation (RobT) involving chromosomes 6D of wheat and 6E^b of Th. bessarabicum (2n = 2x = 14, E^bE^b) through the mechanism of centric breakage–fusion is reported here. An F₂ population was derived from plants double‐monosomic for chromosome 6D and 6E^b from crosses between a DS6E^b(6D) substitution line and bread wheat cultivar ‘Roushan’ (2n = 6x = 42, AABBDD) as female parent. Eighty F₂ genotypes (L1–L80) were screened for chromosome composition. Three PCR‐based Landmark Unique Gene (PLUG) markers specific to chromosomes 6D and 6E^b were used for screening the F₂ plants. One plant with a T6E^bS.6DL centric fusion (RobT) was identified. A homozygous translocation line with full fertility was recovered among F₃ families and verified with genomic in situ hybridization (GISH). Grain micronutrient analysis showed that the DS6E^b(6D) substitution line and T6E^bS.6DL stock have higher Fe and Zn contents than the recipient wheat cultivar ‘Roushan’.     

Dissection of a major QTL for seed colour and fibre content in Brassica napus reveals colocalization with candidate genes for phenylpropanoid biosynthesis and flavonoid deposition

A major quantitative trait locus (QTL) influencing seed fibre and colour in Brassica napus was dissected by marker saturation in a doubled haploid (DH) population from the black‐seeded oilseed rape line ‘Express 617’ crossed with a yellow‐seeded B. napus line, ‘1012–98’. The marker at the peak of a sub‐QTL with a strong effect on both seed colour and acid detergent lignin content lay only 4 kb away from a Brassica (H+)‐ATPase gene orthologous to the transparent testa gene AHA10. Near the peak of a second sub‐QTL, we mapped a copy of the key phenylpropanoid biosynthesis gene cinnamyl alcohol dehydrogenase, while another key phenylpropanoid biosynthesis gene, cinnamoyl co‐a reductase 1, was found nearby. In a cross between ‘Express 617’ and another dark‐seeded parent, ‘V8’, Bna.CCR1 was localized in silico near the peak of a corresponding seed fibre QTL, whereas in this case Bna.CAD2/CAD3 lay nearby. Re‐sequencing of the two phenylpropanoid genes via next‐generation amplicon sequencing revealed intragenic rearrangements and functionally relevant allelic variation in the three parents.     

Transgressive segregation for reduced glucosinolate content in Brassica carinata A. Braun

Successful commercial utilization of the meal by‐product of Brassica oilseed crops requires the cultivation of cultivars with low glucosinolate (GSL) content in the seeds; however, such cultivars are not yet available in Brassica carinata. The objective of the present research was to search for transgressive segregants with further‐reduced GSL content in the progeny of crosses involving four B. carinata lines with reduced GSL content (90 compared with 120 μmol/g seed in standard germplasm). The four lines were crossed following a diallel design and F₂ phenotypes (F₃ seed bulked) were analysed for GSL content. F₂ phenotypes with a transgressive GSL content lower than the parents were identified in all crosses involving the line S2–1241, suggesting that this line carries alleles for reduced GSL content not present in the other lines. F_{3 : 4} lines from transgressive F₂ phenotypes were evaluated for 2 years, which resulted in the selection of an F_{3 : 4} line with an average GSL content of 58 and 46 μmol/g seed, respectively compared with 84 and 62 μmol/g seed, respectively in S2–1241.     

Development and characterisation of a <Emphasis Type="Italic">Brassica carinata</Emphasis> inbred line incorporating genes for low glucosinolate content from <Emphasis Type="Italic">B. juncea</Emphasis>

The presence of high levels of sinigrin in the seeds represents a serious constraint for the commercial utilisation of Ethiopian mustard (Brassica carinata A. Braun) meal. The objective of this research was the introgression of genes for low glucosinolate content from B. juncea into B. carinata. BC₁F₁ seed from crosses between double zero B. juncea line Heera and B. carinata line N2-142 was produced. Simultaneous selection for B. carinata phenotype and low glucosinolate content was conducted from BC₁F₂ to BC₁F₄ plant generations. Forty-three BC₁F₄ derived lines were selected and subject to a detailed phenotypic and molecular evaluation to identify lines with low glucosinolate content and genetic proximity to B. carinata. Sixteen phenotypic traits and 80 SSR markers were used. Eight BC₁F₄ derived lines were very close to N2-142 both at the phenotypic and molecular level. Three of them, with average glucosinolate contents from 52 to 61 micromoles g⁻¹, compared to 35 micromoles g⁻¹ for Heera and 86 micromoles g⁻¹ for N2-142, were selected and evaluated in two additional environments, resulting in average glucosinolate contents from 43 to 56 micromoles g⁻¹, compared to 29 micromoles g⁻¹ for Heera and 84 micromoles g⁻¹ for N2-142. The best line (BCH-1773), with a glucosinolate profile made up of sinigrin (>95%) and a chromosome number of 2n = 34, was further evaluated in two environments (field and pots in open-air conditions). Average glucosinolate contents over the four environments included in this research were 42, 31 and 74 micromoles g⁻¹ for BCH-1773, Heera and N2-142, respectively. These are the lowest stable levels of glucosinolates reported so far in B. carinata.     

Marker‐assisted introgression of rare allele of β‐carotene hydroxylase (crtRB1) gene into elite quality protein maize inbred for combining high lysine,tryptophan and provitamin A in maize

Vitamin A deficiency in humans is a widespread health problem. Quality protein maize (QPM) is a popular food rich in lysine and tryptophan, but poor in provitamin A (proA). Here, we report the improvement of an elite QPM inbred, HKI1128Q for proA using marker‐assisted introgression of crtRB1‐favourable allele. HKI1128 was one of the parental lines of three popular hybrids in India and was converted to QPM in our earlier programme. Severe segregation distortion for crtRB1 was observed in BC₁F₁, BC₂F₁ and BC₂F₂. Background selection by 100 SSRs revealed mean recovery of 91.07% recurrent parent genome varying from 88.78% to 93.88%. Across years, introgressed progenies possessed higher mean β‐carotene (BC: 9.22 µg/g), β‐cryptoxanthin (BCX: 3.05 µg/g) and provitamin A (proA: 10.75 µg/g) compared to HKI1128Q (BC: 2.26 µg/g, BCX: 2.26 µg/g and proA: 3.38 µg/g). High concentration of essential amino acids, viz. lysine (mean: 0.303%) and tryptophan (0.080%) in endosperm, was also retained. Multi‐year evaluation showed that introgressed progenies possessed similar grain yield (1,759–1,879 kg/ha) with HKI1128Q (1,778 kg/ha). Introgressed progenies with higher lysine, tryptophan and proA hold immense potential as donors and parents in developing biofortified hybrids.     

Independent selection for seed free tryptophan content and vernalization response in chickpea domestication

Chickpea shows a distinct domestication trajectory vis‐a‐vis pod dehiscence and growth cycle mediated by vernalization insensitivity compared with its companion Near Eastern legumes. Our objectives were: (i) to map the quantitative trait loci (QTLs) associated with vernalization response and seed free tryptophan in domesticated × wild chickpea progeny and (ii) estimate the genetic correlation between vernalization response and free tryptophan content. A domesticated × wild chickpea cross was used to document phenotypic segregation in both traits and to construct a skeletal genetic map for QTL detection. A number of vernalization response and seed free tryptophan content QTLs were documented in both F₂ and F₃ generations. No significant genetic correlation between these two traits was observed. Epistatic relationship between two free tryptophan loci was documented. It is evident that selection for high seed tryptophan is easier to accomplish relative to selection for vernalization insensitivity. This suggests that the two traits were selected independently in antiquity, thereby corroborating earlier claims for conscious selection processes associated with chickpea domestication.