ITMI20070841A1 - MAIS PLANT WITH REDUCED MYCOTOXIN CONTENT AND ITS USE IN FOOD AND EXTRACTIVE FIELD - Google Patents

Description

Description of the patent for industrial invention entitled:

"CORN PLANT WITH A REDUCED CONTENT OF MYCOTOXINS AND ITS USES IN THE FOOD AND EXTRACTIVE FIELD"

The present invention relates to a corn plant with a reduced content of mycotoxins and capable of accumulating considerable quantities of anthocyanins in the tissues. The invention also concerns the use of the plant or its parts in human or animal nutrition and as a starting material for the extraction of antioxidant pigments.

BACKGROUND OF THE INVENTION

Corn is one of the most important crops in the world and quantitatively it is the main one in Italy: about 10% of the harvested product enters directly or indirectly into the human food chain. In Italy, cultivation is particularly intense in the Northern regions (Piedmont, Lombardy, Veneto, Friuli Venezia Giulia, Emilia Romagna) and in the lowland areas of the Center-South, where there is greater water availability. Overall, the cultivation of maize covers an area of about 1.1 million hectares, with an annual production of over 10.6 million tons.

From the end of the 1970s to the end of the 1990s, the productions increased from 50 to more than 100 quintals per hectare, allowing a great development of the livestock sector in particular in the Po Valley, where corn is by far the primary source of calories. in animal feed, in the form of grain, mash and silomais. In fact, the use of corn in Italy is divided up to 82% for livestock use, 12% for starch industries, 4% for direct human use and 2% for industrial purposes. From the processing of corn, in addition to food products, paper, bioplastics and solvents are obtained and increasingly in the future, corn will also be used to obtain energy.

Corn, in the form of grain, flour or silage (deriving from the ear or the whole plant), is one of the most used raw materials for animal nutrition. In fact, it is the main component of the diet for pigs and poultry and also finds important applications in the nutrition of ruminants. The high availability of corn flour has promoted the development of farms in the regions of Northern Italy which, in terms of technical level and product quality, rank at the top of the community landscape.

The presence of mycotoxins in corn grain is a topic that has only recently been taken into serious consideration, despite the now certain relevance for the implications both on the health of farmed animals and for humans. Mycotoxins are substances produced by hygrophilous and hygromesophilic fungi that are able to infect the grain before harvesting. These fungi, mainly belonging to the Penicillium, Aspergillus and Fusarium genera, are capable of producing toxic and carcinogenic metabolites for humans and farmed animals, capable of entering the food chain. The development of these fungi, which are naturally present in virtually every field, can also occur on incorrectly stored grains, thus representing a problem that affects the entire maize chain, from the cultivation, harvesting, drying and storage phases.

Among the numerous mycotoxins, aflatoxins can pass directly to the foods used by humans or pass to the foods produced (eggs, milk, cheese, etc.), through the feed used to feed animals. These substances, toxic to humans and animals, cause serious diseases (1, 2). The problem is particularly felt, since in particular years and / or with incorrect agronomic practices, the thresholds for the accumulation of aflatoxins and / or fumonisins permitted by law may be exceeded, resulting in the loss of the harvest (3, 4). In Northern Italy, where corn is the most important crop, there are favorable climatic conditions for the development of fungi capable of producing fumonisins and, practically every year, the grain is contaminated to a greater or lesser extent depending on the year. Fumonisins are produced by Fusarium verticillioides, found in the crop residues of the fields where corn is grown and able to infect plants in every stage of development in the following season. The problem is particularly pressing, since if the EC regulation 856/2005 is applied by the European Commission, starting from October 2007 the limits set by the community legislation for fumonisin will become 2000 ppb (parts per billion) for the grain, 1000 ppb for flour and 400 ppb for products for direct human consumption. These limits will be particularly penalizing for Italy, where the pedoclimatic conditions favor the accumulation of these mycotoxins, forcing a large part of the crop to be downgraded to other uses such as energy.

The antioxidants contained in plant species destined for the human diet are divided into nutrient compounds (vitamin C and carotenoids) and non-nutrients (flavonoids and phenolic compounds). They are able to prevent cell damage caused by active oxygen species and free radicals that are formed during aerobic metabolism or as a result of exogenous stress factors, and this thanks to their prerogative to oxidize easily and become the preferential target of such active oxygen species. In particular, flavonoids are compounds that determine some of the various pigments that can be observed in the plant world. They are water-soluble molecules, which are found in glycosilated form within the vacuole; based on their chemical structure they are grouped into different classes, of which the most important are flavanones, flavonols, isoflavonoids and anthocyanins. These compounds have a basic structure, consisting of a 15-carbon ring, resulting from a condensation reaction involving phenylalanine and acetate.

Anthocyanins represent a class of secondary metabolites, synthesized exclusively in plants, capable of giving the red color to the tissues in which they are accumulated, as well as performing various other physiological functions (5). Several studies indicate that these molecules possess antioxidant activity and that, if taken in the diet, they can have important health effects on various human and animal diseases, such as cancer and heart diseases (6, 7, 8, 9, 10, 11, 12). In particular, it has been observed that mice fed with colored seed corn varieties are better protected against obesity and hyperglycemia in a high-fat diet (11) or against the onset of cancer induced by carcinogens (7) . It was also highlighted that anthocyanins exert an antimicrobial effect with a significant reduction in the synthesis of aflatoxins in vitro (13).

The corn plant is able to accumulate anthocyanins in different ecotypes but, currently, the hybrids present on the market and used all over the world accumulate negligible quantities both in the plant and in the grain. In corn, the genes involved in the biosynthetic pathway that leads to the accumulation of flavonoid-type pigments are activated by two classes of regulatory genes: the Cl / Pll and Rl / Bl family of genes. The production of pigments in any plant tissue generally requires the interaction of at least one member of each family (14, 15, 16, 17, 18, 19, 20, 21, 22).

Given the great genetic variability existing in this species, several alleles of the regulatory genes of the anthocyanin biosynthetic pathway have been characterized and are known to confer strong pigmentation in various plant tissues: B1 {booster), PII (purple plant), RI {red color), and PI (pericarp color) (14, 15, 23, 24). In particular, the alleles B1 and PII, when present in the same genotype, induce a large accumulation of anthocyanins in various tissues of the plant (roots, stocco, leaves, anthers, cob, pericarp and, partially in the leaves) giving an intense red color; RI confers the accumulation of anthocyanin pigments in the aleurone and PI confers the accumulation of phlobaphenes in the grain. These alleles are dominant and able to act in a single dose, lending themselves well at the genetic level for the constitution of hybrid seeds.

Industrially, anthocyanins are extracted from the skin of red grapes, as a by-product of the wine industry. The extraction takes place with diluted acids and, by drying, a water-soluble powder relatively rich in these pigments is obtained. Given the high cost required by the extraction operation, currently on an industrial level the anthocyanin pigments are not extracted from any species rich in these pigments, such as the American blueberry (900-4500 mg / kg), blackberry and currant black (600-3500 mg / kg) as their unit yield is very low.

The interest aroused in recent years by various studies concerning the beneficial effects of anthocyanins on various human diseases such as cancer and heart diseases, encourages us to identify plant sources that are able to accumulate large quantities, easily extractable at industrial level and at low costs. , to be added to human and animal diets (6, 7, 8, 9, 10, 11, 12, 24, 25).

DESCRIPTION OF THE INVENTION

It has now surprisingly been found that maize plants having a genotype selected from the following: (B1 - PII- RI- PI- C1-); (B1 - PI- Cl-); (. R - PI-Cl); (Snl - PII- RI- PI- C1-); (Snl - PI- C1-), where the capital letter indicates the dominant allele of the gene concerned and the trait of any dominant or recessive allele of the same gene, have a significantly reduced accumulation of mycotoxins compared to maize plants with a different genotype. The same combinations of genes also determine a significant increase in antioxidant pigments in the plant tissues, in particular anthocyanins and phlobaphenes.

Therefore, in a first aspect the invention refers to a corn plant, including the grain generated by it, having a genotype chosen from those specified above, where:

- B1 indicates the booster 1 gene, also known as colored planes 1, located on chromosome 2 (bin 2.03); Genbank Accession #: X70790; SEQ ID NO: 1 (see the attached List of Sequences);

- PII indicates the purple plant 1 gene, located on chromosome 6, bin 6.04; Genbank Accession #: AF015268; SEQ ID NO: 2;

- PI indicates the pericarp color 1 gene, located on chromosome 1, bin 1.03; Genbank Accession #: M73028; SEQ ID NO: 3;

- RI indicates the red color 1 gene, located on chromosome 10, bin 10.06; Genbank Accession #: AF135456; SEQ ID NO: 4

- CI indicates the colored aleurone 1 gene, located on chromosome 9, bin 9.01; Genbank Accession #: AF320613; SEQ ID NO: 5;

- Snl indicates the scutellar node color 1 gene, located on chromosome 10, bin 10.06; Genbank Accession #: X60706; SEQ ID NO: 6.

The genes B1 {booster 1), PII {purple plant 1) and, to a lesser extent, Snl {scutellar node color) determine the accumulation of anthocyanin pigments in the pericarp; RI {red color 1) and CI {colored aleuronel) favor the accumulation of anthocyanins in the aleurone while PI {pericarp color 1) increases the amount of phlobaphenes in the pericarp (Fig. 1). The hybrids {Bl- PII- RI- PI- Cl), the result of the crossing between the lines constituted B1B1 PllPll C1C1 and R1R1 PIPI C1C1, and the equivalent {Snl- PII- RI- PI- C1-) are able to accumulate pigments in all the tissues of the plant and are therefore particularly preferred. The genes Bl and PII or Snl and PII, when present in the same genotype, favor the accumulation of anthocyanins as well as in the grain, in practically all the tissues of the plant (Fig. 3). The analysis of the anthocyanins contained in the grain of the RI and Bl PII genotypes showed an accumulation respectively of 123 and 234 times higher than the control genotype bearing the ri gene (Fig. 4). It has been seen that the presence of these pigments in the grain hinders the formation of mycotoxins in the open field. In fact, the analyzes carried out on the mycotoxin content in the grain showed a lower accumulation of Fumonisin B1 in the genotypes capable of accumulating pigment in the seeds, in particular in the pericarp with the PI and B1 PII genotypes, compared to the isogenic controls that differ only for the capacity to accumulate pigments (Fig. 5). The aflatoxins Bl, B2, Gl and G2 were found to be below the 0.01 ppb threshold in all the samples analyzed.

The corn plant having the desired genotypic combination can be obtained by crossing lines appropriately selected based on the phenotypic characters, using classical breeding methods. Figure 2 shows a diagram of the crosses necessary to pyramid different genes in a single genotype. The crossings were made with paper bags both for covering the ears, to avoid contamination of exogenous pollen, and for collecting pollen from the plumes.

In another aspect, the invention relates to the use of a maize plant described here, or a part thereof, as a food intended for human or animal consumption. The high content of antioxidant pigments, combined with low mycotoxin contamination, makes the food use of the corn plant according to the invention particularly attractive and advantageous. In a preferred embodiment, the plant is used to produce grain, mash or chopped for human or animal nutrition.

In a further aspect, the invention relates to the use of the corn plant described here, or a part thereof, as a starting material for the extraction of antioxidant pigments, in particular anthocyanins. The extraction processes are known to the expert in the field and include, for example, extraction with solvents, such as methanol and acetone, and extraction with supercritical C02. A technique that can be used for the extraction of pigments from the corn plant is described in EP1 191071.

The invention is illustrated in greater detail by the attached figures and the subsequent experimental part.

DESCRIPTION OF THE FIGURES FIG. 1. Comparison of the pigment accumulation in the grain in some of the possible genotypic combinations that can be constituted through targeted crosses between the genes B1 {booster 1), PII (purple plant 1), RI {red color 1), CI (colored aleurone 1) , PI (pericarp color 1) and corresponding recessive alleles.

FIG. 2. Diagram of the crossings carried out for the constitution of the two parental lines B1B1 PllPll and R1R1 P1P1, which can be directly used as parental for the constitution of the hybrids Blbl Pllpll and RI ri P1p1 or crossed between them to form the hybrid Blbl Pllpll Rlrl P1p1.

FIG. 3. Comparison of the accumulation of anthocyanins in the adult plant, in the chopped and in the grain between two hybrids different in the genetic constitution only for the presence of the B1 and PII genes. Comparisons made in the 2005 and 2006 seasons.

FIG. 4. Analysis of the anthocyanin content accumulated in the grain in the ri, RI, Bl, PII genotypes in the 2006 season.

FIG. 5. Analysis of the content of Fumonisins in the grain in the RI, PI, Bl PII genotypes and in the respective isogenic controls ri, pi and bl pii. Analyzes carried out in the 2005 and 2006 seasons.

EXPERIMENTAL PART

The various lines and hybrids produced were obtained by manual pollination by bagging the inflorescences to avoid contamination by exogenous pollen.

Quantitative determination of anthocyanins

The extraction of anthocyanins was carried out on aliquots of flour obtained from dry grains. The extraction is carried out in a mortar kept on ice, given the sensitivity of the pigments to degradation, using quartz sand and a fixed volume of the extraction buffer containing 95% ethanol and 1% HC1. The homogenate is centrifuged for 10 minutes at 13000 rpm, the sumatant is recovered and the precipitate is deprived of all traces of pigment by repeating the extraction operation several times. The total sumatant is used for the quantitative determination of anthocyanins, by spectrophotometer at 530 nm, expressed as mg of cyanidin-3-glycoside equivalent (molar extinction coefficient 26900 L m <-1> mol <-1> and PM = 449, 2) per 100 g of grain.

Mycotoxin analysis

The mycotoxin analyzes were carried out by the Granaria Association of Milan, Laboratory of Chemistry and Microbiology on dry grain samples of the different genotypes. In particular, the analyzes were carried out by HPLC for the Anatoxins Bl, B2, Gl and G2, while the analysis on Fumonisin Bl was carried out using the Fluorimetric method.

BIBLIOGRAPHY

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LIST OF SEQUENCES

1) SEQ ID NO: 1

1 actagtgggc gaaatgtaaa tgtccctaga ttttttggag cttggccatt taaccaaatt 61 gagtgccaga aaagagcttt gttacccata cctgctataa aagtcatgga cacacgaaaa 121 agttctttgt ctattttgtttc acaagggatta ccggt tcactgt

181 ttccatttaa accaacatta tattagcctc agtgccctag tgaaacgctc gaggtctagg 241 atatccaatc ctctcaaggc ttttggtgtg cacaccatta attgactaat ttatcaagca 301 gtggccacca ttaaccttat ctggttcttc gcctttccat aggaaacctc tcctatatca 361 gtcaattttt ttgattagcc atttctgagc tgggaaaata gtgagatggt agattggttg 421 cgacgaaaga acagttttca ccaaaatatc gcgacctacc gaggatagga acttcccttt 481 ccaactttac aggcaacatc caattttgtc agttagaggt tgaacgtcaa tcctccttag 541 cttgcgaatg tgcagaggta gacccaaata cttacccaaa aaaattgcaa tcttaatagg 601 gaagcttcgc ctgatttcag ctatcatatt ttatatgcag gggattggga agatctctgt 661 tttgttcata ataaccttca gtccacagaa gaactcaaag tgctaatata tttaattagc 721 aattaggata agagtaaaac gcaaattgag gagctaaaaa aggaggtttg agactggcta 781 ctgggtctat atgacacgca ctttttaaat ttcgagatag ggaataggga acatatatat 841 agtagccata tacaactttg cgtttaagga tgtgtttgat ttgacttttt tctttgactt

901 ttgccctctc aaactcaaaa ggtaaattaa agggcttagt ccagtaagtg gctttttttc 961 aaaagtcgac tttctcacag cataaaacta agggcctgtt tggtttaatg cctaacttgc 1021 aacactttgc ctaacttttg tgtctaaggt tagttcttca attcgaacga ctaaccttag 1081 gcaaagtgtg acacatttag ccacgaacca aacagcccct aaaggcacat ttaaacctat 1141 tttagtggct tttaggtaga actgtctaaa tatatatgga agaattttta gtggttttta 1201 tcaaatgatt tttaactttt tatagaactc acatgccaca atagtttttt ctctcacaaa 1261 aactttttca cggccactgt attactgtat atttacgatc gcacgttacc ttaatagttt 1321 acacaacata ataaatgcgt acgttgctac atgccggagc tagtggtata cttgttgcgt 1381 cgccgtcttg ctcggtcact ttggagctcc gtccacaaac ccatctcgga ttccaaggct 1441 cgtggcatac ctgtagtcat atattcgtcc ccccgttcct cagtagccac cgaggtcacc 1501 attgccacag agagtgtatg tgggtggcta tatatatatg cctgcttgca ttgcacacgt 1561 acgtacagcg tagcggggag cggaggtgag ctcctcctcc gattcttgcc taatctttgg 1621 tctttgcaca cgtacgaaag ctttttgcat tgtttcgttg cttctggatg actggatgat 1681 cagtactcct agatattaag cgataccgat ctagaatcga gttgttgtac tctctctgtc 1741 cct tttgtgc agctataact agctaggttc cttcgcatag agcctctcta cagagtacag 1801 actagctagc agtgtcaggc acgaaatgga aatggctact tccaaattgc acgagctgga 1861 attatatact cttctgatct tcttcttaccgt ctctgatatag ctgg

1921 tcttgcttgc gtgaaggatt agtaagcgcg tgatggcgct ctcagcttgc ccggctcagg 1981 aagaactgct gcagcctgct gggaggccgt tgaggaagca gcttgctgca gccgcgagga 2041 gcatcaactg gagctattcc ctcttctggt ccatttcaag cactcaacga ccacggtaaa 2101 ttgaagtcct gataatctat aatctgtctg gcagttttct acaactctga tgaatgatcc 2161 tcacatcgtt tgcctgatac atacataccg catacatatg aaataaagaa agtcggatcc 2) SEQ ID N0:2

1 aagctttgcc tttcatgtct ttgtgtactc ttggcttgtt agcatgttct agagcttttc

61 caagtgcgat tttttttggc cttgattgat attcttggtt tgaaaagtca tttaaacatc

121 gattttacca atgttgacat tcatcgggag acgtcctaac caatggtttg ccactaaaag 181 atgacaatga cgttcgtttg gaacccactc aaccatctgt ttgcaacgtg ctctatttgg 241 aaaccggagg aagcgactcg ccacctaaaa ttaacgcgag ccattcggat caaacgagct 301 actactccta tatctgacag ttgccaccct cacacatacc gtacgcgtac gatggtgcag 361 ttctcacttg gaagtttgaa ccgtcgtcac tgctccctgc agggaccaga cgacggccag 421 acgacccagc atcaccagca ctgtcgtggt ccgtacgtgc ctgcagttta gcgtttgcag 481 ggtctcaact cggccacgta gcggcagttc tacggagtga agcgaaccga ccagctgggg 541 gcctgggcca cgcacgtcga cagcgtgcat ttaaatacgc cggcggagct cgatcaacga 601 aagtcgatcg agagaccctg ctgctagcta gctggacacc gagagagaaa gagagagaga 661 gcgagagatg ggcaggaggg cgtgctgcgc caaggaaggg gtgaagagag gggcgtggac 721 ggccaaggaa gacgatacct tggccgccta cgtcaaggcc cacggcgaag gcaaatggag 781 ggaggtgccc cagaaagccg gtaaataaat tgttgattag cttttctttc agatttcgtt

841 ttgggatcag catatcatat atatatacca agacaagatc gacgacgacg cggctgatgg 901 tgtgtgtgca ggtttgcgtc ggtgcggcaa gagctgccgg ctgcggtggc tgaactacct 961 ccggcccaac atcaagcgcg gcaacatctc ctacgacgag gaggatctca tcgtccggct 1021 ccacaagctc ctcggcaaca ggttacatga gctgacgacg agcagcccca tctgctacga 1081 attcatccgt tccggtgttg gccgtgagcg agatagagtg atcgagctca tgtttgtcta 1141 ctcctttaca tacatacgta cgtacgtacg tgttggcatg taggtggtcg ctgattgcag 1201 gcaggctgcc gggccgaaca gacaatgaaa tcaagaacta ctggaacagc acgctgggcc 1261 ggagggccgg cgccggcgcc ggcggcagca gggtcgtctt cgcgccggac accggctcgc 1321 acgccactcc ggcggcggcg gggagccacg agatgaccgg cggccagaag ggcgccgctc 1381 ctcgcgcgga cctcggctcg cccggctccg cagcagtagt gtgggcgccc aaggcggcgc 1441 ggtgcacggg cgggctcttc ttccaccggg acacgccgca cgcgggcgag acggagacgc 1501 cgacgccaat gatgatggcc ggtggtggag gaggagaagc acggtcgtcg gacgactgca 1561 gctcggcggc gtcggtatct cccctcgtag gaagcagcca gcacgacccg tgcttctccg 1621 gcgacggcga cggcgactgg atggacgacg tgagggccct ggcgtcgttt ctcgagtccg 1681 acga ggagtg gctccgctgt cacacggccg agcagcttgt gtagacaaca agtacacgta 1741 cggagtacaa ttatatatag ccatgcgtat gtatactttt ttcccttctc caatccaaac 1801 agaaatacgt atgcttgtatga aatagtatatac

3) SEQ ID N0: 3

1 agccagcaca gcacacacac tggaaagtgc aagctgtagt gagacctgcg cgactgccag 61 cgtgtatccg cgcggcaagg agcgtagcgc cggtcgtcgg cccgcacggc caccaactcc 121 cttggacgca cgcgcgacca gctgctaacc gtgcgcaagt agtagtgcga cttcgccgcc 181 ggccgggatc gctagctcga tcgatcggcg ggaccacata cgactccggt gtggccagcg 241 gcggccgggc cggggaacgc acgtgctgcg agcgagcgag ggcagacgct agctgttgcc 301 gggagctagc cggcgcgcga tggggaggac gccgtgctgc gagaaggtgg ggctcaagcg 361 agggaggtgg acggcggaag aggaccagtt acttgccaac tacattgcgg agcacggcga 421 ggggtcctgg aggtcgctgc ccaagaatgc aggcctgctc cggtgcggca agagctgccg 481 gctccggtgg atcaactacc ttcgggcgga cgtcaagagg gggaacatct ccaaggagga 541 agaagacatc atcatcaagc tccacgccac cctcggcaac aggtggtccc tgatcgccag 601 ccacctcccc ggccgaacag acaacgagat caagaactac tggaactcgc acctcagccg 661 gcagatccac acgtaccgcc ggaaatacac cgccgggcct gacgacaccg ccatcgccat 721 cgacatgagc aagctgcaga gcgccgacag gcggcgcggc ggcaggaccc cgggccggcc 781 gccgaaggct agcgccagca ggaccaagca ggcggacgcc gatcagcccg gcggcgaggc 841 gaaaggcccg gccgcggc gg cgtcgagccc gcggcacagc gacgtggtga acccgggccc 901 gaaccagccc aacagcagca gcggcagcac gggcacggcc gaggaggagg ggcccagcag 961 cgaggacgcg agcgggccgt gggtgctgga gccgatagag ctcggggacc tagtctgggg 1021 ggaggccgac agcgagatgg acgccctgat gcctatcggg cccggcggca cgactcggct 1081 gccctcgaag ggcttggcgc ggtcggctgc gaggcccagg tggacgacct gttcgacatg 1141 gactgggatg gcttcgcggc ccatctgtgg ggcgggccgg agcaggacga gcacagcgcg 1201 cagctgcggc aggccgccga gccgctggaa gttgctgctg ctgctgctgc tgcgacggcg 1261 gcccgcaccc cggacgatcg cgagctggag gcgttcgaga cttggctcct gtccgactcg 1321 ttctgacggc tccggtcacc ggaccgatca gacagaccaa ataattgggt cacgtgtgct 1381 cgctcgctcg ctgccgtcgc gtgggtcttg gttcagatgg ccaaataatt gggaaaaaaa 1441 ttctacggcc agggccgtaa agccaccacc gtgcgctcct gatgtcgatg cctgccgcgt 1501 ggagctcttg cgtatctaac gctcccacga caatcaccct tccagacggc tcgaattaca 1561 tacgacagga tcggctccgc tctactccgt tctgttcgct tctgctttag gtgcgtgcct

1621 agcagatggt gaggcggcgt cgcgcggccc tcccgacggc tcgccggccg cgctacgggg 1681 cctgctgcag cagcccctcc tccacgcctg taaaagagct ttgtatttac ctgtttgttttt 1741 aacaatggatgtttgttt 1741 gtgcttgtattgttat gtgcttgtattgattat 180

4) SEQ ID NO: 4

1 ccttgtgttc gcaatctccc ccttgatgag tgcattgtca attacking tatgagtata

61 tgtaaacata aagaggaaga agaagagaaa ctcactcaaa tgaccaaaag ccagataaaa 121 tccatataag tcatttggag taagcaaaac tttgtcccca aagaaaacgg caatgactca 181 acacgactaa gcgagaaaaa ggttagccct caataagagg caaaagtagg gctcaacacc 241 actagcgaga agccaaaatc tcatcggagc acaaaaagct ttattggagc ataaaaacca 301 atccagggca taaaatcagc acaaaagcta aaatcaaagc tagtttctct agaaagaggc 361 aaacaactca acagaactag caaaaatctc caaaaggctc aattctcctc ccctgtcata 421 atactccaaa aaatctctca caaagaccaa catctccaaa taacacatct ctcccccttg 481 gatgagagga agcccgaacg acaaaatttc tcccccttga tagaggaact gggaaatctc 541 aaaatctctc acttttgttg acaagtgacc tcattaggca caaacaaggg tagaaagaaa 601 aacttcccct cgacaagaga agtccccgtg agaaaatgca gaaatgcaac agagagtagg 661 aaatggagaa aatgcaggag cttcaggagt ataccagtca aagtgtaaaa atgttctaag 721 tggtgttgtg ctaagtgtgc atcaataaat gcacgtgcta gcagatgctc aaactgatca 781 tgtgcacaag atatgtgaag tgtaagtatt ttgattcagt tttaaacatt ttaaagaggt 841 gttcatcaca aagatagcac taagcatata ataagtagga aatcaactaa gtgccagtaa 901 caggtaaaca aggtga acta cccaaacaac ggtcactcat gatgctagaa acatatatga 961 aatagatacc atttgaaatt ttcaagattt atcggatgtt gcagcggatt ccaaatttca 1021 ttgaaaggca tgtgagtgtg agaggttgta agcattgtct ttgtctaact tttcaaccgg 1081 atgtagccta tgcagaaaag caaccagaag ctaaaaacac ggtttttatc ttctactaca 1141 ctgctcaagt tcatccaaaa gtataaatgg aagcagtctc gatacagtga tgtggtggca 1201 aatgcaaaca cgtctcgatc ttcctgatta acacttataa cccacgagct ttttgaacca 1261 aacaagggtt gttgtgggtt gaacccgtcc caaccatcat caactcgcta gccaaacaca 1321 cgcttagggg ccaaagcagt gctataatat gagtggtggc gctattatat atagcgtcag 1381 agaacttaga tctgatattc tgatgaagaa aaaatgactt tactgactac gaaagaagaa 1441 gaaaggagct atagagagag aaaaaaagag gggtcgtgta gtgcttaaac tgtacatgaa 1501 cagcagtagt gttacagaag ctaaactcaa ccagagctcc accaaagaca aagagggtct 1561 acttccatca ccgtcttgct cggtcacttg gagctctgtc cataaattaa acccatcttg 1621 gatcccaagg ttcgtggcat atctgtaggc atctaccccg tcttcgtcgt ccgctcctca 1681 ctagctacca agaggtcgcc attattgcca acatagagtg tacgtggatg tctatatata 1741 tgcctacttg cacccatatg gc ataggcgt tcgatcccct tagcgcggag gagagctcct 1801 ccggttcttc tctacccttc gcatggaagt tcttgcattg cttcgttgct tctctagttt

1861 cttccttcta cgtctttcca gcatacgcat gcccctcgtc cgccggttca cgaggcatcg 1921 tctgatgatc agtagataat aagcaatata atactgatct agaatcgagt tgttgtactc 1981 ttcgcagata ggttcgttcc ttcacataga agcgagtaca gactacagac cacacagtat 2041 cagctggcac gaaacgaaaa tggttacttg caaattgcat gcacgagcta gaattatatt 2101 cttctaatct tcttcgttga ctttctggct tcagcaggcg cgtgatggcg ctttcagctt 2161 cccgag

5) SEQ ID N0: 5

1 tctagaccca taatttagtg tttgtttaat tgctggatta tttgcgctag attatataat

61 ctggagagat tataatcaga aacaaacagg gtctaaggct gagcccagca gagttcctat 121 ttggattcgc atacgtaaaa taggtagagt ggctactgtt taccgttcca acagagcccg 181 tatccgtctt cgcaaattgc gatgggctcg cgacgcttcc cttctctgcg ctttctctct 241 cctcccccta aatccaaatc aaacaaccgt cccatagtac ggcggtctcg cccctcttct 301 tttcttggcg ggaggcagcg gcagttgcaa atgtgtgctg gaataagaag agccgctgga 361 gaaaagaaat agataggaag agttttgtgt ggatcatata tatgtactat acctcctaaa 421 ataggaatgt gaatccgata tctactggag tcggtctaaa agccttgttt atcctttccc 481 agtggcgggc ctagaggttg aagggagcat gagcaagaac taaaagcatc tcatattttt 541 tttatatcta gctacaggta tagagccatg cgtgaccaac tcgatggata gctttgtctc 601 ttttagctgt tgtgtacttg tgtcttgtgc cggagtctgg acgcgcaggt gcccagggtc 661 gtcgagccct cggaccgcca ctgtgctttc ctaaattact ttttttcgca gttacagctt 721 cgaacaattt gttgacaaat cctttgtttt cggttgtagt ccaattttgt gtatattttc

781 tcaagtacgg tttctaataa atcgttccca tactatgtgt gcaacgtttt cttatacaca 841 acaattttgc gtatacctat atcatactac attctatcca ttttaggaaa ttaaaaagat 901 aattttgtca accacgcaaa tgtacatcag agttgtctgt tcacacagtt atttgcaatt 961 ttgcataata gtaaaaatac atgacatgat taggtcatgc cagtaagcca aaagccagta 1021 ataacagatt cggagcagca agttcgcaat ataattggcc aaggctagga ttcgtggtcc 1081 atgtatagta cacccttcgt ttttttattt atcgcgattt agtttaaaaa taaaattaac 1141 ttacaacaaa tattcgagaa tatacatagt attatttaaa tattatatta cagttgtctg 1201 gtcacaaaac tagtaaaaaa ggctcacagc ctacggtgcg atatattttt acagatagat 1261 ttgattatca acctcctgtg ttatttttta gtgacggttt cttaaaaaac accactagaa 1321 atcgtatttt tataggtggt tccttaagaa atctgcatgc agaaatccat ggcggttttc 1381 ttaagaaacc gtatgtagaa atacgatttc tagtgacgat cttcttaagg aaaccaccac 1441 taaaaattat ttttatcctt aattttcgag tttttcaatt gacctcgtac gatgaaccta 1501 tcaaaataaa agttgtacat ctctaaaagt tatgaaaatt tgtagttaac aactttttta 1561 tttgaactca ttttggttct caaaaattgc atctaaattt gtcaaattta aaattcaaat 1621 tttcc aaacg acctcggatg aaaaaagtgt caaaatgaaa gttgtagaac ttcaaaagtt 1681 attcaacttt gtagtcgact atctttttat ttgaattcgc ttacggtctc aaacaagcaa 1741 tttacactca gttggttgta atatgtggac aataaaacta caaactagac acaaatcata 1801 ccatagacgg agtggtagca gagggtacgc gcgagggtga gatagaggat tctcctaaaa 1861 taaatgcact ttagatgggt agggtggggt gaggcctctc ctaaaatgaa actcgtttaa 1921 tgtttctaaa aatagttttc actggtgatc cttagttact ggcatgtaaa aatgatgatt

1981 tctactgtct ctcgtatgga cggttataaa aaataccatt atattgaaaa taggtctctg 2041 ctgctacact cgccctcata gcagatcatg catgcacgca tcattcgatc agttttcgtt 2101 ctgatgcagt tttcgataaa tgccaatttt ttaactgcat acgttgccct tgctcagcac 2161 cagcacagca gtgtcgtgtc gtccatgcat gcactttagg tgcagggcct caactcggcc 2221 acgtagttag cgccactgct acagatcgag gcaccggtca gccggccacg cacgtcgacc 2281 gcgcgcgtgc atttaaatac gccgacgacg gagcttgatc gacgagagag cgagcgcgat 2341 ggggaggagg gcgtgttgcg cgaaggaagg cgttaagaga ggggcgtgga cgagcaagga 2401 ggacgatgcc ttggccgcct acgtcaaggc ccatggcgaa ggcaaatgga gggaagtgcc 2461 ccagaaagcc ggtaaaacta gctagtcttt ttatttcatt ttgggatcat atatataccc 2521 ccgaggcaag accggaggac gatcacgtgt gtgggtgcag gtttgcgtcg gtgcggcaag 2581 agctgccggc tgcggtggct gaactacctc cggcccaaca tcaggcgcgg caacatctcc 2641 tacgacgagg aggatctcat catccgcctc cacaggctcc tcggcaacag gtctgtgcag 2701 tggccagtgg tgggctagct tattacacga gctgacgacg aggcgatcga tcgagcgtct 2761 gctgcgaatt catctgttcc ggtgtcggcc gtgtgagagt gagctcattc atatgtacat 2821 g cgtgttggc gcgcaggtgg tcgctgattg caggcaggct gcctggccga acagacaatg 2881 aaatcaagaa ctactggaac agcacgctgg gccggagggc aggcgccggc gccggcgccg 2941 gcggcagctg ggtcgtcgtc gcgccggaca ccggctcgca cgccaccccg gccgcgacgt 3001 cgggcgcctg cgagaccggc cagaatagcg ccgctcatcg cgcggacccc gactcagccg 3061 ggacgacgac gacctcggcg gcggcggtgt gggcgcccaa ggccgtgcgg tgcacgggcg 3121 gactcttctt cttccaccgg gacacgacgc cggcgcacgc gggcgagacg gcgacgccaa 3181 tggccggtgg aggtggagga ggaggaggag aagcagggtc gtcggacgac tgcagctcgg 3241 cggcgtcggt atcgcttcgc gtcggaagcc acgacgagcc gtgcttctcc ggcgacggtg 3301 acggcgactg gatggacgac gtgagggccc tggcgtcgtt tctcgagtcc gacgaggact 3361 ggctccgctg tcagacggcc gggcagcttg cgtagacaac aagtacacgt atagatgtcc 3421 aataagcacg aggcccgcga gcccggcacg aagcccgctt tttgggcccg gtccgagccc 3481 ggcacggccc ggttatatgc agacccgggc cggcccggca cgaataagcg ggccgggctc 3541 ggacaggaaa ttaggcacgg tgagctagcc cggcacggcc cgtttaggtc taagcccgtt 3601 aagcccgttt ttttacacta aaacgtgctt ctcggcccgc atagcccgct tctcggcccg 3661 ctttttt cgt gctaaacggg ccggcccggc ccgtttaggc ccgttgcggg ccgggctcgg 3721 acaggaaatt gagcccgcgt gcttagccgt cccggcccgg ttttttaatc gtgcctggcg 3781 ggccaggccc aaaacgggcc gggcttcacc gggcccgggc cggaccgggc cgggcggccc 3841 gtttggacat ctctaagtac acgtatggag gagaatatat atatagtcat gcgtacgtat 3901 agattttttc atccgatccc aacagaaata cgtatgaaaa tgctcttcgt tctttttcat

3961 ttatcatatc tatactatac ttaaaacacc agtttcaacg gtcgtcatgc gtcatttttt

4021 tacaaataac ccctcacagc tatttcaaat taatccgctg cacgtctata gatgccaaac 4081 gacgcccaac acgggctaga tgcacgcggg ccacaactat ggcacaggca cgtcatgccg 4141 gcctgctaac tgtgtcgggc tagcccgtta gcccgtcgat ccatttaatt aaattagcgt 4201 aacgacgccc gacacgggct agatgcacgt gggccacaac tatggcacat gcacgtcatg 4261 ccggcctgtt aactgtgtcg ggccagtctg ttagcccatt gatccattta attaaatcag 4321 cgtaaaatgt taaaaaacgg tgcagtaggt ggggttcgaa cccataccct gatggaagaa 4381 gggcgggaga cactgggtga aactgtctaa ccagtagaat atcacgctaa gatgttttta 4441 atattgaata taaattgtat ataagcatat aagttttttt gtaaaataaa aaaataatcg 4501 tgtcgggccg ggccatcact actggccgag gctacaaccc aagcacgaca cgacgttctt 4561 ggctcttgca agcattaggt cgtttctgag accatattgg cgcaatggac tacatgatgt 4621 ttggggttgc tgaattgaat ggagcagcaa taatttgtca cactaacagc aaaatgaaag 4681 gttatttgtt ggttttaaac gttagtaatt gctacgaagt agcataattt atatggagcg

4741 catccagttt ttattgatgc ctgactttag caatcactcc atattttgat ctatcttttt

4801 tataagtttg acttcatggg acttatttta gaaacttgat ctcacaaact ttctcttatt

4861 ttgtctctat atgatgaaat tgtgtcattt tataatcttt gttcattcag tcaatcgttg

4921 tgaactctct tctaatcact cacttcatta gttgtgttgt accaagacat atttgcatag 4981 agtaaacaat aacatcagtt agccaaatca aaaaatatat tatacagaga gcggagacaa 5041 tcaaataaaa aatcttgaaa tttttttaat ggatagttta cgtgggtatt gttgtaagcc 5101 gtcgcaacgc acgggcaacc gactagtttt agtttataaa ttaataaacg tacgacaaat 5161 attaagaacg ccacctttcc atgcctacgc gcgcgtgaga cacgaccggg gcacgtcagc 5221 acgtgtgccc ctgttgtata atttatttac tttttaatga ctatgtgctg ttggttgccg

5281 ttggcttcat cgtgttcgta gccatgcata aatccagcgc cgtacatgtc gatagagaat 5341 acttgctctt tttcaacaaa aaaaggggta aactgcacta tgtatatagt tataataata 5401 aaatacatac gatgagggag ttatttaatt taattcatca ttgtaacgtt gtacgcaata 5461 ttagagattt atagatattt attattacta gatgtgcgta ataccttagc tagcgctgaa 5521 ggcttctgta atatccttca acttgtgttt accaaattcc cctcttcagt tgagacaaat 5581 ccacgtgtag ccaggaggga gttatttaat ttactccaca tgtgtgaaca catggttgat 5641 aagtatccat gagtatcggt acgaggagca attgtccaca aaaaaaaaca agttttgatt 5701 ctgaggttag ccatatcgtg agtgtggtag cgtactagtg tgcaatgtct tttagcttga 5761 ctagggatga caatttaacc cgtaaacctg gaactcgatg gatatctgat ccgatggtta 5821 cgggtaacgg tgaagatttt gacctgcggg tatagatggc caataagcat gaggcccgcg 5881 agcccggcac gaagcccgct gtttgagccc ggtccgagcc cggcacgacc cgattctatg 5941 cgggtccggg ctggcccagc acgaataagc gggtcgggct cggacaggaa attaggcacg 6001 acctgtttac ctctaagccc gttaggcccg cttttttgca ctaaaacatg cttaccagcc 6061 cgcttagcgc gctttttggc ccgctttttt cgtgctaaac gggccgggtc ggcctgttta 6121 g gcccgctgc gagccggact cggacaggaa atcgagtccg cgggcttaaa cagcttggcc 6181 cgattttcta accgtgcctg gtgggtcggt ccaaaacggg ccgggcttca ctgggcccgg 6241 gccgggccga gcggcccctt tggccatctc tacctgcggg tgtaacctgc acccgatccg 6301 aagtttcgcg ggtgtggata tgggtttcta tttcaaccac ggttgaccta cactcggccc 6361 gaaatttggt ttttttttat tattttgcac aataatgatt aaggtataat agtaattatt

6421 ttaaataagt aagttggcta atgattcatg aattgttttg ttggtacaac ccgcttgctg 6481 agcacatatt tatatctata ctactatatt aaaaacataa tgtgggcacc tggtgctact 6541 atggcttcac cctccccaca tcaagcataa tgtgggcacc tggtgctagc ttcaccctcc 6601 ccgcatcaag cgcctgcaaa aaatagtgca aaactaggac tcaaacccac acccctctgc 6661 tctaga

6) SEQ ID NO: 6

1 tagcgttcg atccccttag cgcggaggag agctcctccg gttcttctct acccttcgca

61 tggaagttct tgcattgctt cgttgcttct ctagtttctt ccttctacgt ctttccagca

121 tacgcatgcc cctcgtccgc cggttcacga ggcatcgtct gatgatcagt agataataag 181 caatata ctgatctaga atcgagttgt tgtactcttc gcagataggt tcgttccttc

241 acatagaagc gagtacagac tacagaccac acagtatcag ctggcacgaa acgaaaatgg 301 ttacttgcaa attgcatgca cgagctagaa ttatattctt ctaatcttct tcgttgactt

361 tctggcttca gcaggcgcgt gatggcgctt tcagcttccc gagttcagca ggcggaagaa 421 ctgctgcaac gacctgctga gaggcagctg atgaggagcc agcttgctgc agccgccagg 481 agcatcaact ggagctacgc cctcttctgg tccatttcag acactcaacc aggggtgctg 541 acgtggacgg acgggttcta caacggcgag gtgaagacgc ggaagatctc caactccgtg 601 gagctgacat ccgaccagct cgtcatgcag aggagcgacc agctccggga gctctacgag 661 gccctcctgt cgggcgaggg cgaccgccgc gctgcgcctg cgcggccggc cggctctctg 721 tcgccggagg acctcggcga caccgagtgg tactacgtgg tctccatgac ctacgccttc 781 cggccaggcc aagggttgcc cggcaggagt ttcgcgagcg acgagcatgt ctggctgtgc 841 aacgcgcacc tcgccggcag caaagccttc ccccgcgcgc tcctggccaa gagcgcgtcc 901 attcagtcaa tcctctgcat cccggttatg ggcggcgtgc ttgagcttgg tacaactgac 961 acggtgccgg aggccccgga cttggtcagc cgagcaaccg cggctttctg ggagccgcag 1021 tgcccgacgt actcggaaga gccgagctcc agcccgtcag gacgagcaaa cgagaccggc 1081 gaggccgcag cagacgacgg cacgtttgcg ttcgaggaac tcgaccacaa taatggcatg 1141 gacgacatag aggcgatgac cgccgccggg ggacacgggc aggaggagga gctaagacta 1201 agagaagccg to ggccctgtc agacgacgca agcctggagc acatcaccaa ggagatcgag 1261 gagttctaca gcctctgcga cgaaatggac ctgcaggcgc taccactacc gctagaggac 1321 ggctggaccg tggacgcgtc caatttcgag gtcccctgct cttccccgca gccagcgccg 1381 cctccggtgg acagggctac cgctaacgtc gccgccgacg cctcaagggc acccgtctac 1441 ggctctcgcg cgacgagttt catggcttgg acgaggtcct cgcagcagtc gtcgtgctcc 1501 gacgacgcgg cgcccgcagc agtagtgccg gccatcgagg agccgcagag attgctgaag 1561 aaagtggtgg ccggcggcgg tgcttgggag agctgtggcg gcgcgacggg agcagcacag 1621 gaaatgagtg gcactggcac caagaagcac gtcatgtcgg agcgaaagcg acgagagaag 1681 ctcaacgaga tgttcctcgt cctcaagtca ctgcttccgt ccattcacag ggtgaacaaa 1741 gcgtcgatcc tcgccgaaac gatagcctac ctcaaggagc ttcagagaag ggtgcaagag 1801 ctggagtcca gtagggaacc tgcgtcgcgc ccatccgaaa cgacgacaag gctaataaca 1861 aggccctccc gtggcaataa tgagagtgtg aggaaggagg tctgcgcggg ctccaagagg 1921 aagagcccag agctcggcag agacgacgtg gagcgccccc cggtcctcac catggacgcc 1981 ggcaccagca acgtcaccgt caccgtctcg gacaaggacg tgctcctgga ggtgcagtgc 2041 cggtgggagg agctcct gat gacgcgagtg ttcgacgcca tcaagagcct ccatttggac 2101 gtcctctcgg ttcaggcttc agcgccagat ggcttcatgg ggcttaagat acgagctcag 2161 tttgctggct ccggtgccgt cgtgccctgg atgatcagcg aggctcttcg caaagctata 2221 gggaagcggt gaaggggcag ctggaaattt ggacatcgac gggcatggaa ggcttcatgg 2281 gatcgaagca aagatgtatt tcttgtttct ttagataaca gacatgaatc ggacctttat 2341 atcaacaatt atatgggcat gaatacttaa gactccagcc cttaacacgt agaaactcaa 2401 aaaagaagag aggaagctaa agactaagcg taaggtatat ttggaagtaa attattttta 2461 tagtttctaa gcaatctcat ggtttatagg aatactagag tgtttatggc ataaggtgtt 2521 tggttgcatt cataaaacct atattttcaa agtcatagca ttctagatac ca

Claims (8)

CLAIMS 1. Maize plant having a genotype chosen from the following: (Bl- PII- Rl-Pl- Cl -), · (Bl- PI- Cl-); (R- PI- CI); (Snl- PII- RI- PI- C1-); (Snl- PI- C1-), where the capital letter indicates the dominant allele of the gene concerned and the trait any dominant or recessive allele of the same gene, where said gene is: - Bl, gene booster 1, located on chromosome 2 (bin 2.03); SEQ ID NO: l; - PII, purple plant 1 gene, located on chromosome 6, bin 6.04; SEQ ID NO: 2; - PI, pericarp color 1 gene, located on chromosome 1, bin 1.03; SEQ ID NO: 3; - RI, red color 1 gene, located on chromosome 10, bin 10.06; SEQ ID NO: 4; - Cl, gene colored aleurone 1, located on chromosome 9, bin 9.01; SEQ ID NO: 5; - Snl, gene scutellar node color 1, located on chromosome 10, bin 10.06, SEQ ID NO: 6. 2. Corn plant according to claim 1, wherein said genotype is selected between (Bl-PII-RI-PI-Cl) and (Snl-PII-RI-PI-C1-). 3. Seed of a corn plant according to claims 1 and 2. 4. Use of a corn plant according to claims 1 and 2, or a part thereof, for the preparation of food intended for human or animal consumption. Use according to claim 4, in the form of grain, mash or chopped. 6. Use of a corn plant according to claims 1 and 2, as a starting material for the extraction of antioxidant pigments. 7. Use according to claim 6, for the extraction of anthocyanins intended for human or animal nutrition. 8. Food intended for human or animal consumption containing a corn plant according to claims 1 and 2 or a part thereof.