2007 Mercedes Intermedio Erucico

Inheritance of high oleic acid content in the seed oil ofmutant Ethiopian mustard lines and its relationship

with erucic acid content

M. DEL RIO-CELESTINO 1*, R. FONT 2 AND A. DE HARO-BAILON 2

1 I.F.A.P.A. Centro Alameda del Obispo (Junta de Andaluca), Alameda del Obispo s/n, 14080 Cordoba, Spain2 Instituto de Agricultura Sostenible (CSIC), Apartado 4084, 14080 Cordoba, Spain

(Revised MS received 29 December 2006; First published online 5 March 2007)

SUMMARY

Ethiopian mustard (Brassica carinata) genotypes with dierent contents of oleic acid (C18:1) in theseed oil could be useful for food and industrial applications. The objectives of the present researchwere to study the inheritance of high C18:1 in the seed oil of dierent lines of Ethiopian mustard andits relationship with erucic acid content (C22:1). The low C18:1/high C22:1 mutant line L-1806, thehigh C18:1/high C22:1 mutant line L-482, the high C18:1/low C22:1 mutant line L-2890 and thelow C18:1/very high C22:1 mutant line L-1630 were isolated after a chemical mutagen treatment ofC-101 seeds (about 94 g C18:1/kg and 450 g C22:1/kg). The high C18:1/zero C22:1 line L-25X-1 wasobtained by interspecic crosses of Ethiopian mustard with rapeseed and Indian mustard. Plants oflines L-2890rC-101, L-482rL-2890, L-1630rL-25X-1, L-1630rL-2890 and L-482rL-1806 werereciprocally crossed and F2 and the BC1F1 generations were obtained. Cytoplasmic eects werenot observed in any of the crosses. The segregation pattern in F2 and BC1F1 populations dieredin the crosses studied. The inheritance of C18:1 content in crosses segregating for this fatty acidwas that expected for one (crosses between L-482rL-1806), two (L-2890rC-101) or three(L-1630rL-2890, L-1630rL-25X-1 and L-482r2890) loci. Oleic acid segregation indicated controlof accumulation by two segregating genetic systems, one acting on chain elongation from C18:1 toC22:1 and the other involving desaturation from C18:1 to linoleic acid (C18:2). Accumulationsof C18:1 and C22:1 were inuenced by the same loci (M1, M2, E1 and E2), which control thechain elongation steps leading from C18:1 to C22:1. In addition, C18:1 was inuenced by oneadditional locus (tentatively named OL) involved in control of desaturation of C18:1 to form C18:2.The genetic constitution of the parent lines would be OlOlE1E1E2E2m1m1m2m2 for L-2890,OlOlE1E1E2E2M1M1M2M2 for C-101, ololE1E1E2E2M1M1M2M2 for L-1630, OlOle1e1e2e2M1M1M2M2for L-25X-1, ol1ol1E1E1E2E2M1M1M2M2 for L-482 and Ol1Ol1E1E1E2E2M1M1M2M2 for L-1806.Transgressive recombinants were obtained from the cross L-1630rL-25X-1, with about three-foldincrease of the C18:1 content of the parents (>643 g/kg) and free of C22:1 content, which represent ahigh potential for commercial exploitation.

INTRODUCTION

The fatty acid composition of Brassica oil determinesits physical and chemical properties. The seed oilof standard Ethiopian mustard (Brassica carinataA. Braun) is characterized by a high proportion oferucic (C22:1), linoleic (C18:2) and linolenic (C18:3)

acids, which together account for about 0.75 of thetotal oil fatty acids. The remaining 0.25 correspondsto the fatty acids palmitic (C16:0), stearic (C18:0),oleic (C18:1) and eicosenoic (C20:0) acids (Getinetet al. 1997).The use of mutagenesis permitted the development

of Ethiopian mustard lines with specic fatty acidproles, such as lines with a high C18:1 content(L-482 with >200 g/kg; Velasco et al. 1997), with alow C18:1 content (L-1806 with

et al. 1997), and lines with low and high C22:1 con-tents whose genetics have been reported in previousstudies (Del Ro et al. 2003) (L-2890 and L-1630 withabout 100 and>500 g/kg, respectively; Velasco et al.1997, 1998; De Haro et al. 2001). These oils withspecic fatty acid proles are in demand because oftheir improved nutritional and/or technologicalproperties (Kinney 1994).Vegetable oils with a high content of C18:1 are

of interest for nutritional and industrial purposes.The reduction of the levels of polyunsaturated fattyacids and their substitution for the monounsaturatedC18:1 is an important goal for the development ofhigher quality mustard oil (Scarth & McVetty 1999).The high C18:1 oil can be heated to a higher tem-perature without smoking, so that the cooking time isreduced and food absorbs less oil (Miller et al. 1987).A diet containing a high content of C18:1 can reducethe content of the undesirable low-density lipoproteincholesterol in blood plasma (Grundy 1986) andmonounsaturated fatty acids more eectively preventarteriosclerosis than polyunsaturated fatty acids(Chang & Huang 1998). Therefore, eorts have beenmade to develop high C18:1 and zero C22:1 geno-types of B. carinata using dierent strategies (inter-specic crossing, mutagenesis, etc.).Nevertheless, their usefulness for commercial ex-

ploitation will depend on the adequate integration ofthe genes controlling the altered biosynthetic pathwayinto inbred lines with a high potential to developagronomically acceptable hybrids. This requires priorknowledge on how both traits (C18:1 and C22:1) areinherited (Takagi & Rahman 1996).Dierent segregation patterns were found in

Brassica napus depending on the populations used forthe genetic study of the C18:1. These genetic studiesindicated that the high C18:1 trait was controlledby one locus (Rucker & Robbelen 1997; Schierholtet al. 2001), two loci (Schierholt et al. 2001) and fourloci (Chen & Beversdorf 1990). Chen & Beversdorf(1990) reported the inheritance of C18:1 and C22:1in three homozygous microspore-derived populationsof spring rapeseed (B. napus L.) ; in the study, ac-cumulations of C22:1 and C18:1 were inuenced bythe same two loci, which control the chain elongationsteps leading from C18:1 to C22:1. C18:1 was fur-ther inuenced by at least two additional segregatingloci involved in control of desaturation of C18:1 toform C18:2.Limited genetic studies have been conducted

to date on the inheritance of the C18:1 content inthe seed oil of Ethiopian mustard. Velasco et al.(2003a, b) reported mono and digenic control forC18:1 content but the relationships and interactionbetween the C18:1 and C22:1 pathways was notinvestigated in those studies.In B. carinata, Del Ro et al. (2003) and Del Ro-

Celestino et al. (2005) reported digenic control of

C22:1 content in the low-C22:1 mutant L-2890, thehigh-C22:1 mutant L-1630 and the line zero-C22:1L-25X-1. The C22:1 content in L-2890 is controlledby partially recessive alleles (m1 and m2) at two lociM1 andM2, in L-1630 is controlled by alleles (E1 andE2) with additive eects at the loci E1 and E2, whileline L-25X-1 was controlled by alleles (e1 and e2) act-ing in an additive and equal manner at two loci E1and E2.Molecular and biochemical studies in rapeseed, a

relative of Ethiopian mustard, have shown that thezero C22:1 concentration is associated with an ab-sence of b-ketoacyl-CoA (KCS) activity (Roscoe et al.2001). KCS, the enzyme that catalyses the elongationpathway of C18:1 to C22:1, is encoded by FAE1genes, which have been shown to be closely linkedto the E1 and E2 loci controlling C22:1 content inrapeseed (Barret et al. 1998; Fourmann et al. 1998).Recent surveys have shown that quantitative traitloci (QTL) for C22:1 content lie in regions of theB. napus genome aligned with the long arm ofArabidopsis thaliana chromosome 4. This alignment isconsistent with a Brassica FAE1 homologue as acandidate gene for the control of C22:1 biosynthesis(Qiu et al. 2006).The objective of the present study was to determine

the genetic control of C18:1 content and its geneticrelationships with C22:1 content in mutant lines(L-482, L-1806, L-1630 and L-2890) and the L-25X-1line of Ethiopian mustard.

MATERIALS AND METHODS

Plant material

The lines used in the present study were the zero-C22:1 line L-25X-1, obtained from interspeciccrosses of B. carinata line C-101 and zero C22:1Brassica juncea and B. napus (Fernandez-Martnezet al. 2001) and the resulting mutants, all obtainedafter treatment of the line C-101 (with low-C18:1 andstandard C22:1 content) with ethylmethane sulpho-nate (Velasco et al. 1995, 1997, 1998): the high-C18:1mutant L-482 and the low-C18:1 mutant L-1806,both with standard C22:1 concentration; the high-C18:1 and low-C22:1 mutant L-2890 and the low-C18:1 and high-C22:1 mutant L-1630. The fatty acidcomposition of these lines is presented in Table 1.

Genetic study

Reciprocal crosses were made between: (i) mutantL-2890 and the lines C-101, L-1630 and L-482 tostudy the genetic control of high-C18:1, both in lowand high C22:1 backgrounds, (ii) between L-25X-1and L-1630, to study the genetic control of high-C18:1 both in zero and very high C22:1 back-grounds, and (iii) between L-482 and L-1806 to clarify

354 M. DEL R IO -CELEST INO, R. FONT AND A. DE HARO-BAIL O N

the relationship between the high and low C18:1,both in high C22:1 background.The seeds of lines L-482, L-1806, L-2890, L-1630,

L-25X-1 and C-101 were analysed for fatty acidcomposition by the half-seed technique (Thies 1971),germinated in a growth chamber and transplantedinto pots at the four true leaf stage. The plants weregrown in the glasshouse at 22/18 xC (day/night) withan 18 h day length, in Cordoba, Spain (37x51k42aN,04x48k00aW; 220 m asl). Crossing was achievedthrough the emasculation of immature ower budsof the female parent followed by immediate polli-nation of its stigmas with fresh pollen from themale parent. Selng was achieved by pollinatingimmature ower buds with pollen from open owersof the same plant. Crosses as well as selfed buds werecovered with paper bags to prevent any contami-nation with external pollen. The fatty acid compo-sition of F1 half-seeds from each cross was analysedby gasliquid chromatography (GLC). The parentsand F1 half-seed from each reciprocal cross weregrown in the glasshouse in winter 1995 (crossesL-25X-1rL-1630, L-1630rL-2890, L-482rL-2890and L-482rL-1806) and in winter 1996 the crossL-2890rC-101. F1 plants were self-pollinated toproduce F2 seed and reciprocally backcrossed to bothparents to obtain BC1F1 seed. Reciprocal crossesbetween the two parents were repeated to obtain F1

seeds in the same environment as F2 and BC1F1 seeds.An evaluation of the fatty acid composition at theF1 plant level was made by averaging the GLCanalyses of the F2 seeds from each individual F1plant. Fatty acid composition was determined froma total of 317 individual F2 seeds and a total of 180BC1F1 seeds from the backcrosses to both parentsof the cross L-2890rC-101, 578 F2 seeds of the crossL-482rL-2890, 580 F2 seeds and 855 BC1F1 seedsof the cross L-2890rL-1630, 749 F2 seeds and 605BC1F1 seeds of the cross L-25X-1rL-1630 and 562 F2seeds and 838 BC1F1 seeds of the cross L-482rL-1806.

Statistical analyses

Means of the C18:1 content were calculated in theparental and F1 generations and compared using theStudents t-test. Since the results did not reveal anymaternal eects for the C18:1 content, the fatty acidcomposition of segregating generations was analysedon single seeds. The C18:1 content of BC1F1, F2 seedswas assigned to phenotypic classes on the basis of theappearance of discontinuities in the scatter plot andthe values found in the parents grown under the sameenvironmental conditions. The proportion of seedsobserved in each phenotype class was compared tothose expected on the basis of appropriate genetic

Table 1. MeanS.E. of fatty acid composition of seed oil of Ethiopian mustard (B. carinata) lines L-2890,L-1630, L-25X-1, L-482, L-1806 and C-101

Lines

Numberof singleplantsanalysed

Number ofhalf-seedsanalyseswithin

each plant

Fatty acids (g/kg oil)*

C18:1 C22:1 C20:1 C18:2 C18:3 C16:0 C18:0

L-2890 (mutantline derivedfrom C-101)

3 50 1962.1 1231.5 1051.3 281.2.5 2063.1 510.6 140.1

L-1630 (mutantline derivedfrom C-101)

3 75 1101.8 5482.6 561.0 781.8 1652.4 250.2 30.1

L-25X-1 (linederived frominterspeciccrosses)

3 50 2494.2 0.40.1 110.4 4596.0 1983.9 611.2 130.4

L-482 (mutantline derivedfrom C-101)

3 63 1621.8 4582.9 840.9 991.9 1452.2 260.4 60.1

L-1806 (mutantline derivedfrom C-101)

3 80 1071.9 4013.3 641.0 2842.1 541.4 390.9 80.3

C-101 (parentalline)

3 25 941.8 4515.0 531.8 2327.4 1114.6 281.4 50.2

* Not including minor fatty acids: myristic, arachidic, palmitoleic, eicosadienoic and nervonic acids.

Oleic and erucic acid in mutant Ethiopian mustard 355

hypotheses. The goodness of t to tested ratios wasmeasured by the chi-square (x2 ) test. Heterogeneity x2

for families within a cross was not signicant(P>0.05) ; therefore, data for families for the samecross were pooled for analysis.

Fatty acid analyses

Fatty acid methyl esters were obtained as describedby Garces & Mancha (1993) and analysed on aPerkin-Elmer Autosystem gasliquid chromatograph(Perkin-Elmer Corporation, Norwalk, CT, USA)equipped with a ame ionization detector (FID) anda 2 m column containing 3% SP-2310 and 2%SP-2300 on a 100/120 mesh chromasorb WAW sup-port (Supelco Incorporated, Bellefonte, USA). Theoven, injector and FID were held at 195, 275 and250 xC, respectively.

RESULTS

Genetic control of high-C18:1, in both lowand high C22:1 backgrounds

Cross between L-2890 and C-101

The fatty acid composition of the seed oil of mutantline L-2890, standard line C-101, and their reciprocalF1s, all grown in the same environment, is presentedin Table 1. The inheritance of low C22:1 content inthe mutant L-2890 was reported in a previous studyand it was concluded that this trait was controlled bytwo loci (M1 and M2) with partially recessive alleles

m1 and m2 (Del Ro et al. 2003; Del Ro-Celestinoet al. 2005).F1 half-seed crosses between L-2890 and C-101

showed a C18:1 mean value of 125 g/kg (Table 2).This C18:1 value was lower than that of the mid-parent (215 g/kg) and dierent from both parents,indicating a partial dominance of low over highC18:1 levels. The reciprocal F1 seeds diered signi-cantly for the C18:1 content (Table 2), indicating theexistence of a slight maternal eect. Since no signi-cant cytoplasmic eects could be detected on theC18:1 content in any of the crosses, the data fromreciprocal F1, F2 and BC1F1 seeds were combinedin Fig. 1.A bimodal distribution pattern was apparent

when the C18:1 data of the F2 generation fromcrosses between L-2890 and C-101 were plotted inscatter plots (Fig. 1d). The rst class was assigned tothe low+intermediate category (C18:1160 g/kg)corresponding to the range observed for L-2890.The number of seeds in each class (Fig. 1d) ttedsatisfactorily to 15:1 ratios (low+intermediate :high)in the ve F2 populations analysed (Table 3) (x

2=3.6,P=0.06). This segregation can be explained as twoindependent loci, controlling the high C18:1 trait.The C18:1 distribution in the BC1F1 backcrosses

to standard line C-101 ranged from 56 to 136 g/kg(Fig. 1b). The distribution was continuous and itwas not possible to separate low and intermediateC18:1 classes ; therefore classes were not tted at any

Table 2. MeanS.E. of oleic acid content of the parents L-2890, L-1630, L-482, L-1806, L-25X-1 and C-101 andof reciprocal F1 at the seed and plant level from their crosses

Generation Parent or cross nC18:1

(g/kg oil) Parent or cross nC18:1

(g/kg oil) P

Parent seed L-2890 50 3363.4 C-101 25 941.8

ratio. The C18:1 content in the backcrosses to themutant line L-2890 (high C18:1) ranged from 74 to344 g/kg (Fig. 1c). The distribution was separatedinto two clear-cut classes, which consisted of seedswith200 g/kg C18:1. A theoreticalgenetic ratio of 3:1 (x2=0.47, P=0.49) for these

classes was expected according to an F2 proportion of15:1. These data indicate that the C18:1 contentin the mutant line L-2890 is controlled by recessivealleles at two loci.Correlations among specic acid content indicated,

in the F2 populations analysed, that C18:1 waspositively correlated with C18:2 content (r=0.81,P

Therefore, the results suggest the existence of allelesat three loci controlling the C18:1 content.Within the F2 population having levels of C22:1

lower than 120 g/kg, C18:1 and C18:2 were nega-tively correlated (r=x0.91, P

population (749 F2), 11.7 seeds for the very highC18:1/zero C22:1 class were expected for both traits,C18:1 and C22:1, segregating independently (P=0.05). In the six F2 populations analysed (Fig. 4d),three from each reciprocal cross, the proportion of 16individuals observed in the very high C18:1/zeroC22:1 class tted satisfactorily a 1:64 ratio (1/4 veryhigh-C18:1/zero-C22:1r1/16 zero-C22:1), which

indicated that the C18:1 content is controlled bythree independent loci (x2=1.35, P=0.24).In the 12 backcrosses to the L-25X-1 parental,

combined in Fig. 4b, two classes were distinguished,one ranging from 81.1 to 300 g/kg and includinglow+intermediate C18:1/intermediate+high C22.1phenotypes, and the second including individualswith high C18:1/zero C22:1 content (300475.4 g

C22:

1 (g

/kg)

600500400300200100

0

L-2890

L-1630

F1

(a)

C22:

1 (g

/kg)

600500400300200100

0

BC1F1 to L-1630 (b)

C22:

1 (g

/kg)

600500400300200100

0

BC1F1 to L-2890 (c)

C22:

1 (g

/kg)

600500400300200100

0

F2(d )

0 100 200 300 400C18:1 (g/kg)

Fig. 3. (a) Scatter plots of C18:1 v. C22:1 concentration(g/kg) in the Ethiopian mustard lines L-2890 (P1), L-1630(P2), and their F1, (b) BC to L-1630, (c) BC to L-2890 and(d) F2 seeds.

C22:

1 (g/

kg)

700600500400300200100

0L-25X-1

L-1630

F1

(a)

C22:

1 (g/

kg)

700600500400300200100

0

(b)BC1F1 to L-25X-1

C22:

1 (g/

kg)

700600500400300200100

0

BC1F1 to L-1630 (c)

C22:

1 (g/

kg)

700600500400300200100

0

0 100 200 300 400 500 600 700C18:1 (g/kg)

F2(d )

Fig. 4. (a) Scatter plots of C18:1 v. C22:1 concentration(g/kg) in the Ethiopian mustard lines L-25X-1 (P1), L-1630(P2), and their F1, (b) BC to L-25X-1, (c) BC to L-1630 and(d) F2 seeds.

360 M. DEL R IO -CELEST INO, R. FONT AND A. DE HARO-BAIL O N

C18:1/kg). The frequencies observed in each class(Fig. 4b) tted satisfactorily a 7:1 ratio (x2=0.63,P=0.42). This segregation would be expected ac-cording to the three-loci model proposed (Table 5).The C18:1 content of the backcrosses to the lineL-1630 seeds showed a continuous distribution(Fig. 4c) and no further division into subclasses waspossible.Within the F2 population free of C22:1 and C20:1

acids, the levels of C18:1 and C18:2 were negativelycorrelated (r=x0.78, P

the maternal eects but no cytoplasmic eects forC18:1 content observed in the present study aresimilar to ndings previously reported in rapeseed byThomas & Kondra (1973) and Rakow & McGregor(1973) and in Ethiopian mustard by Velasco et al.(2003b).There was partial dominance of low over higher

C18:1 content in all crosses except for the crossL-482rL-1806, which indicated additive eects in-uencing seed C18:1 contents (Table 2). The results

obtained in the present study regarding the partialdominance of low over high C18:1 content arein agreement with ndings previously reported inB. carinata (Velasco et al. 2003a, b) but contrastingwith those reported in rapeseed by Rakow &McGregor (1973) and Kondra & Thomas (1975), whoindicated partial dominance for high C18:1/lowC18:2. Similarly to the results obtained in the crossL-482rL-1806, Ecker & Yaniv (1993) reported ad-ditive eects for loci controlling the C18:1 contentand partial dominance of low over high C18:1 con-tent in Sinapis alba.The segregation pattern in F2 and BC1F1 popu-

lations diered in the crosses studied. The inheritanceof C18:1 content in crosses segregating for thisfatty acid was as expected for one (cross L-482rL-1806), two (cross L-2890rC-101) or three (crossesL-1630rL-2890, L-1630rL-25X-1 and L-482r2890) loci. The genetic systems, of one and two locidetermining C18:1 content has been described pre-viously in B. carinata (Velasco et al. 2003a, b) andin B. napus (Schierholt et al. 2001), but dier fromthe model of three loci described in the presentwork (crosses L-1630rL-2890, L-1630rL-25X-1and L-482r2890).Chen & Beversdorf (1990) found dierent segre-

gation patterns in crosses among three homozygousmicrospore-derived populations of spring rapeseed(B. napus) with contrasting amounts of C18:1, whichwere attributed to the existence of two gene systems,each containing two loci. One of these two gene sys-tems has a major eect on C18:1 content which in-volved the co-segregation of C22:1. The other genesystem which aects C18:1 is of minor importance incomparison to the major gene system and was con-trolled by two loci. In the present study, segregationfor C18:1 was also controlled by at least two genesystems. One of these two gene systems has a majoreect on both C18:1 and C22:1 contents, as the seg-regation of these two acids is interdependent; highC18:1 content was always associated with low C22:1and vice versa.The strong negative correlation between C18:1

and C22:1 content in crosses L-2890rC-101,L-1630rL-25X-1, L-482rL-2890 and L-2890rL-1630 (r=x0.93, x0.67, x0.78 and x0.66, re-spectively, all with P

and secondly, the locus OL identied in the presentwork (involved in control of desaturation of C18:1to form C18:2), the genetic constitution of theparent lines would be OlOlE1E1E2E2m1m1m2m2 forL-2890, OlOlE1E1E2E2M1M1M2M2 for C-101, ololE1-E1E2E2M1M1M2M2 for L-1630, OlOle1e1e2e2M1-M1M2M2 for L-25X-1, ol1ol1E1E1E2E2M1M1M2M2for L-482 and Ol1Ol1E1E1E2E2M1M1M2M2 for L-1806.Genetic recombination of loci present in lines

L-1630 and L-25X-1 resulted in transgressive recom-binants with a high potential to develop agronomi-cally acceptable hybrids for commercial exploitation,with about a three-fold increase of the C18:1 contentof the parents and free of C22:1 content. This novelEthiopian mustard germplasm has a linoleic acidcontent of about 180 g/kg. The reduction of linolenicacid content is seen as one of the key requirementsfor improving the value of zero C22:1 oils for fryingapplications (McVetty & Scarth 2002). This lineis hypothesized to have the genotype olol e1e1e2e2M1M1M2M2. The genetic relationship of the olmutant alleles in L-1630 to ol1 alleles in L-482controlling high C18:1 content is unknown. An ad-ditional increase of C18:1 content in recombinantline could be expected by mutagenesis or by in-trogressing the ol1 alleles from the L-482 mutant,if ol1 alleles were at a dierent locus than ol alleles inL-1630.

Because of the low number of genes involved in thegenetic control of the high oleic acid trait in mutantlines L-482 and L-1630, a successful transference ofthis character into breeding lines can be performedin a few generations. The information provided bythe present research will facilitate the developmentof commercial hybrids producing oil rich in oleicacid and free of erucic acid, thus satisfying the in-creasing demand for vegetable oils with high mono-unsaturated fatty acids for nutritional and industrialpurposes. Further molecular studies using popu-lations derived from crosses performed in this workcould be useful for developing a linkage map, basedon these populations, primarily using convenientPCR-based markers that enable integration with ex-isting Brassica linkage maps (Qiu et al. 2006) andwith the A. thaliana genome sequence (ArabidopsisGenome Initiative 2000; Hobbs et al. 2004). With theadvent of molecular markers and the development ofQTL mapping procedures, the selection eciency inEthiopian mustard improvement will be increasedand also could be used to investigate the organizationand regulation of genes responsible for oleic anderucic acid contents in this species.

We thank Alberto Merino and Gloria Fernandez-Marn for their technical assistance. This work wassupported by the M.C.Y.T. (Proj. AGL2001-2293)of the Spanish Government.

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