POLYMORPHISM OF GENES CONTROLLING LOW LEVEL OF LINOLENIC ACID IN LINES FROM VIR FLAX GENETIC COLLECTION

ПОЛИМОРФИЗМ ГЕНОВ, КОНТРОЛИРУЮЩИХ НИЗКОЕ СОДЕРЖАНИЕ ЛИНОЛЕНОВОЙ КИСЛОТЫ, У ЛИНИЙ ГЕНЕТИЧЕСКОЙ КОЛЛЕКЦИИ ЛЬНА ВИР © E.A. Пороховинова 1, Т.В.Шеленга 1, Т.В. Матвеева 2, А.В.Павлов 1, Е.А. Григорьева 1, Н.Б. Брач 1 1 Федеральный исследовательский центр «Всероссийский институт генетических ресурсов растений им. Н.И. Вавилова (ВИР)», Санкт-Петербург; 2 ФГБОУ ВО «Санкт-Петербургский государственный университет», Санкт-Петербург


INTRODUCTION
Areas of flax oil in the world amount to 2.8 million ha.The Russia is the main producer of this culture (709,000 ha in 2016), as the European Union, Brazil, and Japan prohibited the import of transgenic flax grown in Canada [1].However, in flax productivity, Russia is behind Canada, the United States, and France [2].In the Russia, flax ranks fourth in profitability among the oil plants after sunflower, soy, and rape plant and can serve as a reserve plant to compensate for the deficit of the raw material in case of poor harvest of sunflower and soy not only in the South but also in any other regions of the Russia [3].
Flax CVs are split into high linolenic (HL) and low linolenic (LL) according to their purpose.There are 33 CVs in the Russia, and 4 of them are LL [5].
HL flax oil is indispensable for pharmaceutical and perfumery industries, soap production, and the paintwork industry [6].It is critical for the oil to dry fast for the production of paints and drying oils, which is connected to its unsaturation as evaluated by the iodine value (IOD).Normally, it fluctuates in the range from 180 to 190 [7].
When using flax as a major food in case of celiac disease and other alimentary diseases for the replacement of cereal flour, it is recommended to follow the ω6/ω3 acid ratio of 5-10/1 for usual food and 3-5/1 for dietary therapy.The major CVs have a ratio of ~0.25/1 [8]; therefore, to produce full-fat flour, LIN should be reduced.
The first LL cv Linola (Linola TM ) were developed in the 1970s in Canada [9].They contained approximately 2 % LIN and are double-recessive homozygotes in complementary genes ln1 and ln2.The other CVs are successors of cv Linola and were obtained independently from it.They all are patented and unavailable for scientific studies.
Genes fad3a and fad3b (also known as ln1 and ln2) are also considered valuable for flax selection, as they code desaturase that converts LIO into LIN, which is required for processed oil but not desirable for food oil [15].Genes fad3a and fad3b have the size of 3280 and 3002 bp, respectively, and contain 6 exons and 5 introns, each coding proteins with a length of 391 and 392 amino acids.The similarity of genes at the DNA level amounts to 85 %, and the similarity of amino acids amounts to 94 %.Genes differ in dimensions due to indels from 1 to 29 bp localized in introns.Six isoforms are known for fad3a, four of which do not result in ferment inactivation.The first one, A (8 alleles with synonym replacements), is the most widely spread, and isoforms B, C, and E (per 1-2 alleles) are rare.Isoforms D and E lost functionality due to nonsense mutations.Seven isoforms are known for fad3b, five of which do not result in ferment inactivation.These are isoforms A (four alleles), D (eight alleles), G (seven alleles), and E and F (two alleles each).Only two isoforms do not produce full-scale products: isoform B due to nonsense mutation in the first exon and isoform С due to the replacement of histidine with tyrosine in the first His box of the active center of desaturase [11].
Thus, selection modification of the FA content (FAC) of flax oil should be based on the search of spontaneous or induced mutations in genes fad3a and fad3b, the assessment of different combinations of alleles and genes, and the mandatory biochemical confirmation of the oil content modification.
The yellow color of seeds is important for the production of flax of food value.It is controlled by several genes as follows: s1, an inhibitor of anthocyanin specifies white 2019;17 (2) eISSN 2411-9202 Генетические основы эволюции экосистем deformed flower besides the yellow color of anthers and seeds; pf -ad controls the pink color of petals besides the yellow color of seeds; f e determines the light blue corolla and spotted seeds; and ora1 determines the speckled seeds and orange anthers.Three organo-specific genes are responsible for light coloration of seeds, i.e., YSED1 and ysed2 (yellow) and rs1 (light yellow-brown) [16].
A lot of LL CVs were developed worldwide; however, it is not clear how changes of oil FAC affect the other traits.The authors of most CVs do not focus on the LL gene donors as well as on the methods of their selection and verification of the purity of variety.Therefore, the objectives are as follows: (1) identification of the polymorphism of oil seed FAC among the lines and CVs of flax, (2) examination of the link systems between FAC, plant height, and early ripeness of flax group contrast in LIN level, (3) development of CAPS markers for alleles of LL genes, and (4) verification of homozygosis of LL lines created blindly.

MATERIALS AND METHODS
To identify the polymorphism by FAC, 19 HL, 8 medium linolenic (ML), and 16 LL lines were studied (Table 1).Line gc-2 from the accession of old selection (l-1 from k-48) with red-brown seeds was used as the "wild type" (Fig. 1).
Several lines were obtained from each of the two LL cv Linola and cv Eyre; they have different habits and differ in the level of LIN and the FA ratio.Some lines were obtained as a result of crossing of LL line gc-391 (l-2-1 from i-601679, Eyre, Australia) homozygous by genes YSED1 and sfbs1 (inhibition of anthocyanin synthesis in hypocotyls and flowers) with HL lines carrying other genes of the light color of seeds or genes of the economic traits and the further selection of stable forms but without FAC control.Among HL parents, gc-65 (l-3 from k-3178, Tver Province) is homozygous by gene ora1, early ripening, and two lines are obtained from it (HL and ML); gc-109 (l-3 2 from k-6099, Argentina) is homozygous by gene wf1 (white petals) connected to early blossom [17], and five lines are obtained from it (one HL, two ML, and two LL); gc-121 (l-1-1 from k-6272, Northern Ireland) is homozygous by gene rs1 and is involved in the development of two lines (HL and ML); gc-173 is homozygous by gene ysed2 and is involved in the development of two HL lines; and gc-392 (gc-132 × gc-103) is homozygous by genes s1 and sfbs1, and LL line was selected from its hybrid.
From 1995 to 2016, the lines were assessed for the duration of phases of vegetation period (VP) based on VIR Pushkin laboratories: germination-flowering of the first flower (T1), flowering of first flower-ripening of first     Генетические основы эволюции экосистем boll (T2), and germination-ripening of first boll (Т3); the total (Ho) and technical (Ht) height and length of the inflorescense (Inf) were also taken into account.As the weather conditions in the years of examination were different, data were balanced by means of the method of reduced average values [18] to the early ripening standard.
Then the average value for the entire period of examination was calculated for each line.
Oil FAC was assessed by means of gas liquid chromatography with mass spectrometry of the methyl ethers of FA using chromatograph Agilent 6850.The percent content of five FAs in the oil was determined: PAL, STE, OLE, LIO, and LIN as well as the LIO/LIN ratio.
The major characteristic of technical oil is its IOD.This is an indicator of FA oil unsaturation; the higher the IOD, the faster the oil dries.IOD was calculated according to the following formula (AOCS Method Cd 1c-85, cit.ex [11]): where IOD is iodine value of oil and OLE, LIO, and LIN are the shares of oleic, linoleic, and linolenic acids in the oil, respectively.
Data were analyzed using programs Statistica 7.0 for Windows and the analysis package Excel 2007 for Windows [19,20].
The interval for the minimum and maximum values of each indicator was calculated as lim ± НСР (the least significant difference, by Fischer) [21].
To define the effect of the flax group by LIN (HL, ML, and LL) on the plant height, early ripening, and oil FAC, one-way analysis of variance (ANOVA) was used.The share of factor effect (η 2 , %) was calculated according to Fischer [21].
The difference between the flax grouped by LIN level was evaluated by the criterion of Tukey's honestly significant difference for unequal selections with post hoc pairwise comparison of means after declining hypothesis Н0 about the absence of differences with the results of ANOVA [19,20].
The analysis of correlation systems between traits was conducted for each flax group (as per Pearson) plotting correlation pleiade.Factors of correlation between the z-converted matrix of correlation were calculated for the assessment of the similarity of link systems for each of three groups.These traits demonstrate a similarity of matrix structure [22].
DNA was extracted from ten 2-day roots of flax seedling according to the standard technique for each line [23].
A polymerase chain reaction was conducted in the volume of 20 μl.The mixture included DreamTaq™ Green Master Mix (2X) (Thermo Scientific), 5 pmol for each primer, and 1 μl DNA preparation.Primers were synthesized by "Eurogen" [24].Primers flanking the area with supposed mutation in LuFAD3A gene were selected using the program idtdna.com[25].Previously published primers and the protocol were used for the detection of mutations in LuFAD3B gene [13].
DNA was sequenced based on RC of SpbSU "Development of molecular and cellular technologies" and "Eurogen."Reference sequences for gene LuFAD3A used in the work were HM991829.

GENETIC BASIS OF ECOSYSTEMS EVOLUTION
Gene sequences were aligned using the program tcoffee [26].Nucleotide sequences were analyzed using the programs MEGA 7.0.21[27] and UGENE1.290[28].

RESULTS AND DISCUSSION
Using six generations of inbreeding from 26 heterogenic accessions of flax, 40 lines were obtained, of which 19 were HL, 7 were ML, and 14 were LL.
The most early ripening HL lines were gc-2 and gc-65, which had short three VPs (T1 = 39, T2 = 25, and T3 = 64 days) and for late ripening was HL gc-393, a mutant from LL line of cv Linola (T1 = 55, T2 = 48, and T3 = 103 days).HL gc-448 (40 days) and ML l-2 from gc-391 × gc-65 (54 days) were distinguished only for the duration of the germination-flowering period.With the results of ANOVA, no differences were detected between flax groups as per T1 and T3; however, significant differences by T2 were detected.On the other hand,
The content of OLE in oil varied from 9 % (HL, l-1 from gc-391 × gc-173) to 34 % (ML, gc-119).ANOVA demonstrated significant differences for flax groups in this trait stipulated by differences in 7 % between ML and HL by Tukey's test (see Table 2; Fig. 2).
The share of effect of flax group (η 2 ) on the above-described traits with significant differences varied from 17 % to 25 %.For flax groups connected to LIN level in all traits, ANOVA demonstrated significant differences, and the share of effect varied from 77 % to 95 %, which was caused by the concept of division into groups (see Fig. 2).
The LIO/LIN ratio varied from 0.17 to 49.33 (HL, 0.17-0.45;ML, 0.44-3.46;and LL, 9.07-49.33).Minimum values were demonstrated by all HL flax, maximum LL.The LIO/LIN ratio is a strongly varied trait (CV 149 %).However, in HL form, it is more permanent (CV 27 %) than in ML (CV 92 %) and LL (CV 44 %).ANOVA demonstrated significant differences for flax groups by this trait (see Table 2); in terms of Tukey's test, only LL had a significant difference from HL and ML forms (see Fig. 2).
Only the oil of l-2 from gc-391 × gc-109 of all studied accessions can be used for nutritional therapy "as it is"; for dietary therapy, two lines of CVs gc-391 and gc-523 as well as hybrid lines l-4 from gc-391 × gc-109 can be used.The rest of the oils require blending.
FAC in seeds sometimes correlates with VP phases and heights.As described previously [10], changes in the seeds in the reduction of LIN synthesis cause nonsymmetric changes in the FA ratio, which affects the correlation between them.In all three groups of flax, two pleiades of correlated traits were detected.Only a few correlations coincided in each of them: in the first one, heights are closely connected to each other as Ht amounts to approximately 90 % of Ho; in the center of the second one, the content of LIN in oil negatively correlates with the LIO/LIN ratio and positively correlates with IOD.Similar to the first case, this is the result of arithmetic regularities of the calculation of the last two traits (Fig. 3).
In HL lines, the first pleiade is formed with duration of VP (T3) closely positively correlated to its phases (T1 and T2) as well as moderately positively with PAL, formed at the beginning of the biosynthesis of FA.T2 negatively correlates with Ht, which in turn is very strongly positively connected to Ho.The second pleiade, besides its very strong positive correlation with LIN and IOD and strong negative correlation with the LIO/LIN ratio as well as its strong negative correlation between the latter ones, has a strong negative correlation of OLE with LIN and IOD as well as a moderate negative correlation of latter ones with STE.LIO is strongly positively connected to the LIO/LIN ratio and moderately negatively connected to LIN.FAs negatively correlated with LIN are its precursor.Inf is independent from any other traits (see Fig. 3).
The group of ML lines is small in number, the correlation between its traits is significant at r > 0.75, and many strong and all moderate correlations cannot be taken into account.The first pleiade is formed by T1, besides the heights, which is strongly positively connected to Ht; they both negatively correlate with T2.The content of OLE strongly negatively correlates with the first two traits.The second pleiade is formed with positively strongly correlated in pairs LIO and IOD as well as with LIO and the LIO/LIN ratio.These two groups negatively correlate with each other.Inf, T3, and the content of saturated acids do not depend on any other traits (see Fig. 3).

GENETIC BASIS OF ECOSYSTEMS EVOLUTION
In LL lines, the first pleiade is formed with heights, and T1 and Inf are positively connected to each other.T1 positively correlates with T3, and T2 is positively correlated to it.The center of the second pleiade is shifted for IOD, which is positively connected to LIO (strongly) and LIN (moderately) and negatively with OLE, STE, and PAL.LIO also strongly negatively correlates with OLE and moderately with STE; the latter one in turn is positively connected to PAL.LIN very strongly negatively correlates with the LIO/LIN ratio as the variability of LIN determines the variance of this ratio.Pleiades are connected to one another with moderate negative correlation of OLE with T1 and Inf; the latter has positive correlation with IOD (see Fig. 3).
Correlations between the z-converted matrix of correlations for flax groups demonstrated their moderate similarity (r HL-ML © =© 0.50, r HL-LL © =© 0.42, and r ML-LL © =© 0.54), which is mostly stipulated with arithmetic regularities of trait measurement/calculation.
Using factor analysis (principle components method), two major factors were detected that affect the examined traits (Fig. 4).
The first factor determines the LIO/LIN ratio.It indicates antagonism LIN, IOD, and the heights of plants (Ho and Ht) on the one hand and the LIO, PAL, and the LIO/LIN ratio on the other hand.It characterizes approximately 40 % of the total variance.This factor with small overlap separated LL lines from HL. ML lines took intermediate position.It is interesting that overlapping toward LL is caused by the availability of ML heterozygosis in selection (l-2 from gc-391 × gc-109) as well as revertant to HL from LL CV (gc-472).
The second factor determines early ripening and demonstrates antagonism to duration of all three phases of VP on the one hand and OLE on the other hand.It explains approximately 20 % of the total variability.This factor distinguished early ripening, as a rule, high oleic gc-2, gc-65, and l-1, l-2, l-4, l-5 from gc-391 × gc-109, and experimentally late ripening low oleic gc-393 as well as late ripening low oleic gc-483, gc-472, l-1 and l-2 from gc-391 × gc-173.
Thus, factor analysis allows grouping lines in correlated traits and characterizing them in complex.
A new method of identification of alleles of LuFAD3A gene was developed for the search of differences between lines LL and ML by genes of biosynthesis of LIN, which controls its formation.An analysis of available NCBI sequences of alleles of LuFAD3A gene demonstrated that in most LL accessions, the mutation-inhibiting synthesis of LIN is in the first exon of the gene.Primers FAD3Ae1F (acttggcatcctgcattactt) and FAD3Ae1R (ccagaaagataatgtgaaattacc) were constructed that fix this area.The product of amplification has a length of 526 bp.When it is sequenced, each of mutant allele LuFAD3A of lines gc-391 and gc-515 similar replacements is detected in position 28 (G → A), position 255 (G → A), and position 309 (A → G) of the first exon, which indicates the identity of alleles between one another and the full similarity with sequence from the NCBI for genotype SP2047 (HM881831).The replacement in posi- The use of restrictase HaeIII allows to identify the mutant allele containing replacement G 255 → A 255 .Two sites of restriction are available within the amplified fragment of the allele (Fig. 5).When it is decomposed, fragments 85, 90, and 351 bp are formed.The mutation specified above is in position 442 of the product of amplification and affects one of the two sites of restriction, which results in the formation of fragments 90 and 436 (351 + 85) bp.Thus, we proposed a new option of CAPS marker.It helps to identify that CVs ЛМ98 and Istok, as well as lines from LL cv Linola (gc-390 and gc -523), Eyre (gc-391, gc -441, and gc-420), Walaga (gc-395), Amon (gc-474), and accessions 852, 853, 854, 858, and 864 (gc-512-gc-516), are homozygous in mutation in this area of gene (Table 3).
For the second gene, LuFAD3B, the previously published protocol was used for the detection of mutation in the first exon [13].The product of amplification has a length of 468 bp.When it is sequenced, the mutant alleles of lines gc-391 and gc-515 detected replacement in position 6 (С → T) of the second exon, which resulted in the replacement of Hys → Tyr.It was shown that the use of restrictase BsaJI allows to identify the mutant allele.Two sites are available for restrictase within the amplified fragment of allele of the wild type (Fig. 5).Fragments with a length of 191, 240, and 37 bp are formed

GENETIC BASIS OF ECOSYSTEMS EVOLUTION
during its decomposition.The mutations specified above are in position 431 of the product of amplification and involve one of the three restriction sites, which results in the formation of fragments with a length of 191 and 277 (240 + 37) bp.A restriction product of 37 bp of HL forms is combined with the low molecular nonspecific products of amplification; the one gets false impression about the difference of HL and LL forms in size of the larger product of amplification.Thus, the CAPS marker proposed previously for genotypes 593 to 708 can be used for gc-391.It was detected that lines from LL CVs Linola (gc-394), Eyre (gc-391, 420), and Walaga (gc-395) and accession 858 (gc-515) are homozygous by mutation in this area of gene (Table 4).Based on the available data of the NCBI sequences of allele of LuFAD3B gene (KF026416), it was detected that cv Amon has the same point mutation as gc-391.LL cv Eyre (Australia) was one of the first to come to VIR colllection that was heterogeneous by LL genes.Several lines were obtained on its basis, but only one line is used for crossing (gc-391).By means of obtained markers, homozygosis of hybrids from cross-breeding of LL (gc-391) and HL lines (see Table 3; Fig. 5) by gene LuFAD3A was confirmed.Progeny F 7 l-1, 1-3, 1-4 from hybrid gc-391 × gc-109, l-2 from hybrid gc-391 × gc-65 and progeny F 9 l-1 from hybrid gc-391 × gc-392 are homozygous by recessive allele of LuFAD3A gene.Progeny F 9 l-  Although the references describe only two genes responsible for LIN synthesis, according to our information, the genetic surrounding is rather important.The LL CVs available for us are late ripening and are not adapted to the conditions of Russia; therefore, the study objective was to select prospective early ripening forms.It was shown that hybrids, in which breeding record the line gc-109 presented, were ripen 8 to 10 days earlier than the parental LL line gc-391.The most prospective ML line l-3 and LL lines l-1 and l-4 were from the crossing of gc-391 × gc-109.

CONCLUSION
Lines of VIR flax genetic collections have a wide variety of FA oil.A sharp reduction of the synthesis of LIN causes nonsymmetric changes in the FA ratio, which effects the correlation between them and other traits.Factor analysis clearly splits the lines by the level of LIN and the traits connected to it.Primers were constructed, restrictases were selected, and an experiment protocol was developed for the identification of alleles of LuFAD3A gene.It was determined that all LL forms available in the VIR collection have mutation in the first exon (G 255 × A 255 ) of this gene.The test system [13] developed for mutation identification in the first exon of LuFAD3B gene of genotypes 593 to 708 can be used for mutation in the second exon of gc-391, etc.
Four generations of inbreeding are sufficient in most cases for the selection of homozygous forms.

Fig. 1 .
Fig. 1.Pedigree of hybrid origine lines involved in the study

Note:
Ho -the total height; Ht -the technical height; Inf -the length of the inflorescence; T1 -the duration of the phase of germination -flowering of the first flower; T2 -the duration the phase flowering of the first flower -ripening of the first boll; T3 -the duration of the phase of the germination -ripenting of the first boll; PAL -palmitic acid; STE -stearic acid; OLE -oleic acid; LIO -linoleic acid; LIN -linolenic acid; IOD -iodine number; Minimum values selected by Tukey HSD criterion are indicated in italics maximum values are indicated in bold ТОМ 17 № 2 2019 ISSN 1811-0932

Fig. 2 .
Fig. 2. The proportion of influence (η2) of the linolenic acid degree (HL -high linolenic, ML -medium linolenic, LL -low-linolenic) and random variation (error) according to the results of one-way analysis of variance (ANOVA).* differences between groups are significant; Ho -the total height; Ht -the technical height; Inf -the length of the inflorescence; T1 -the duration of the phase of germination -flowering of the first flower; T2 -the duration the phase flowering of the first flower -ripening of the first boll; T3 -the duration of the phase of the germination -ripenting of the first boll; PAL -palmitic acid; STE -stearic acid; OLE -oleic acid; LIO -linoleic acid; LIN -linolenic acid; IOD -iodine number; F is the Fisher criterion value; p is the probability of similarity of classes (HL, ML, LL); differences -significant differences of these classes according to the results of a posteriori comparison by the Tukey criterion for unequal sampling

Fig. 3 .
Fig. 3. Correlation pleiades of traits of pland height, duration of phases of vegetative period and the fatty acid composition of seed oil of flax accessions, differing in the level of linolenic acid synthesis: a -high linolenic; b -medium linolenic; c -low linolenic.Ho -the total height; Htthe technical height; Inf -the length of the inflorescence; T1 -the duration of the phase of germination -flowering of the first flower; T2 -the duration the phase flowering of the first flower -ripening of the first boll; T3 -the duration of the phase of the germination -ripenting of the first boll; PAL -palmitic acid; STE -stearic acid; OLE -oleic acid; LIO -linoleic acid; LIN -linolenic acid; IOD -iodine number

Fig. 4 .
Fig. 4. Factor loading for 13 studied traits (а) and factor scores for 40 lines of flax (b) in the system of two factors.Ho -the total height; Ht -the technical height; Inf -the length of the inflorescence; T1 -the duration of the phase of germination -flowering of the first flower; T2 -the duration the phase flowering of the first flower -ripening of the first boll; T3 -the duration of the phase of the germination -ripenting of the first boll; PAL -palmitic acid; STE -stearic acid; OLE -oleic acid; LIO -linoleic acid; LIN -linolenic acid; IOD -iodine number .HL -high linolenic, SL -medium linolenic, NL -low-linolenic

Table 3 Polymorphism©of©flax©lines'©LuFAD3A gene©according©to©the©length©of©restriction©fragments©(CAPS©markers)
Генетические основы эволюции экосистем of this gene.Progeny F 7 l-2 from hybrid gc-391 × gc-109 appeared to be heterozygous by this gene.Line l-2 from gc-391 × gc-121 is interesting: it is homozygous by the recessive allele of LL gene with the results of DNA analysis, but it has the boundary content of LIN; therefore, it was attributed to HL instead of ML.Thus, parental line gc-121 has a different way to slightly increase the level of LIN.