阅读说明：本技术 一种利用抗赤霉病种质sf7el9改良小麦赤霉病抗性的育种方法 (Breeding method for improving wheat scab resistance by using scab resistant germplasm SF7EL9 ) 是由 戴毅 陈建民 李海凤 石俊涛 高勇 于 2021-08-03 设计创作，主要内容包括：本发明涉及一种利用抗赤霉病种质SF7EL9改良小麦赤霉病抗性的育种方法,1个7EL特异性分子标记Th7EL-46可有效地鉴定出是否含有长穗偃麦草7EL染色体片段,便于分子标记辅助选择育种。利用该标记扩增的产物经凝胶电泳后,450bp的条带为7EL染色体片段特异带。另外,结合荧光原位杂交鉴定可以有效地区分易位染色体是纯合还是杂合,如果只含有1条易位染色体,表明材料为杂合易位系；若含有2条FISH核型一样的易位染色体,表明材料为纯合易位系。主要是通过远缘杂交手段,利用抗赤霉病小麦-长穗偃麦草7EL小片段易位系SF7EL9与栽培小麦杂交,并在杂交后代中选育出抗赤霉病的小麦种质。本发明提出的Th7E-46可作为生物标志物应用于实时鉴定抗病种质中含有的长穗偃麦草7EL染色体片段。(The invention relates to a breeding method for improving wheat scab resistance by utilizing a scab resistant germplasm SF7EL9, 1 7EL specific molecular marker Th7EL-46 can effectively identify whether a thinopyrum elongatum 7EL chromosome segment is contained, and molecular marker-assisted selective breeding is facilitated. After the product amplified by the marker is subjected to gel electrophoresis, a 450bp band is a 7EL chromosome fragment specific band. In addition, the identification combined with fluorescence in situ hybridization can effectively distinguish whether the translocation chromosome is homozygous or heterozygous, and if only 1 translocation chromosome is contained, the material is a heterozygous translocation line; if the material contains 2 translocation chromosomes with the same FISH karyotype, the material is a homozygous translocation line. Mainly utilizes a distant hybridization means to hybridize the scab resistant wheat-elytrigia elongata 7EL small fragment translocation line SF7EL9 with cultivated wheat, and breeds the scab resistant wheat germplasm in the hybridization offspring. The Th7E-46 provided by the invention can be used as a biomarker to be applied to real-time identification of the elytrigia elongata 7EL chromosome segment contained in disease-resistant germplasm.)

1. A breeding method for improving wheat scab resistance by using a scab resistant germplasm SF7EL9 is characterized by comprising the following steps:

step 1), taking the scab resistant germplasm SF7EL9 as a parent, namely a male parent or a female parent, hybridizing with a variety with better high yield in breeding, and F₁Backcrossing the generation or hybridizing with another variety with better yield to obtain BC₁F₁100 seeds;

step 2), planting BC by single plant₁F₁In the seedling stage, molecular marker Th7EL is used-46 pairs of BC₁F₁Selecting plants, reserving plants containing the Th7EL-46 marker, and harvesting seeds from the plants to obtain BC₁F₂Seeds;

step 3), planting BC by single plant₁F₂Selecting by using molecular marker Th7EL-46 and genome fluorescence in situ hybridization GISH at seedling stage, and obtaining seeds of plants containing Th7EL-46 marker and translocation chromosome by dividing the plants to obtain BC₁F₃Seeds;

step 4), planting BC by single plant₁F₃Line, every 10 BC₁F₃The strain is planted with a contrast parent 1 and a parent 2, wherein the parent 1 is SF7EL9 or Yangmai 23 or Annong 8455, and the parent 2 is Yangmai 23 or SF7EL9 Annong 8455; selecting plants containing Th7EL-46 marker and translocation chromosome in seedling stage, selecting plants with plant height, plant type and gibberellic disease resistance superior to those of control parent in grain filling stage and yellow falling stage, and harvesting seeds to obtain BC₁F₄Seeds;

step 5), planting BC by single plant₁F₄Line, every 10 BC₁F₄The strain is planted with a contrast parent 1 and a parent 2, wherein the parent 1 is SF7EL9 or Yangmai 23 or Annong 8455, and the parent 2 is Yangmai 23 or SF7EL9 Annong 8455; selecting plants containing Th7EL-46 marker and a pair of translocation chromosomes in seedling stage, selecting plant lines with plant height, plant type and gibberellic disease resistance superior to those of control parents in grain filling stage and yellow falling stage, and harvesting seeds by single plant to obtain BC₁F₅Seeds;

step 6), planting BC by single plant₁F₅Line, every 10 BC₁F₅The strain is planted with a contrast parent 1 and a parent 2, wherein the parent 1 is SF7EL9 or Yangmai 23 or Annong 8455, and the parent 2 is Yangmai 23 or SF7EL9 Annong 8455; selecting plants containing Th7EL-46 marker and a pair of translocation chromosomes in seedling stage, selecting plant lines with plant height, plant type and gibberellic disease resistance superior to those of control parents in grain filling stage and yellow falling stage, and harvesting seeds in a mixed manner to obtain BC₁F₆Seeds;

step 7), planting in different districtsBC₁F₆Seeds and three cells, and simultaneously planting a control parent 1 and a parent 2, wherein the parent 1 is SF7EL9 or Yangmai 23 or Annong 8455, and the parent 2 is Yangmai 23 or SF7EL9 Annong 8455; identification of gibberellic disease resistance at flowering time, Mixed harvest of BC₁F₆And (3) drying the seeds, weighing, and keeping the yield to be obviously higher than that of the control parent 1 and the parent 2, so as to obtain the new scab resistant wheat strain with high yield.

2. The breeding method for improving the scab resistance of wheat by using the scab resistant germplasm SF7EL9, according to claim 1, wherein 1 specific marker Th7EL-46 is used for tracking the thinopyrum elongatum 7EL chromosome segment in the new scab resistant wheat line, and the molecular marker primers are Th7 EL-46F: TTACACTAACCCATGGTGTT, Th7 EL-46R: GCAGAGAATGAAGCAAAATC, the primer is used to perform PCR amplification on the filial generation line, a specific band of 7EL 450bp can be amplified, and the marker can effectively trace the 7EL translocation chromosome.

Technical Field

The invention relates to a breeding method for improving wheat scab resistance by utilizing a scab resistant germplasm SF7EL9, belonging to the technical field of plant breeding.

Background

Wheat scab (FHB) is a major worldwide disease caused by pathogenic bacteria such as Fusarium asiaticum and Fusarium graminearum, and seriously affects wheat yield and quality. After wheat is infected with gibberellic disease, besides the yield is obviously reduced, toxin Deoxynivalenol (DON) is accumulated in grains, and great hidden danger is brought to food safety. With the change of global climate warming and cultivation system and mode, the growing area of wheat scab in China has a tendency of continuously expanding, the growing area of wheat scab in 2005 + 2010 is about 6196 ten thousand mu, the growing area of wheat scab in 2011 + 2018 is increased to 8632 ten thousand mu, and the growing area of wheat scab in 2019 + 2020 is increased to about 9000 ten thousand mu. How to prevent and control gibberellic disease is a major challenge facing wheat production at present. The breeding of disease-resistant variety by using wheat scab-resistant gene is one of the most economic and effective ways to control the damage of wheat scab. However, wheat is relatively deficient in genetic resources for resisting gibberellic disease, and only 4 sites are defined as wheat scab resistance genes (Fhb1, Fhb2, Fhb4 and Fhb5) at present and are used in wheat genetic breeding. The scab resistant gene is also found in wheat closely related species, such as Fhb3 from grass galileo, Fhb6 from Japanese elymus japonicus, and Fhb7 from decaploid elytrigia elongata. Therefore, the gene for resisting the gibberellic disease in the wheat kindred species is transferred into the wheat, which is beneficial to enriching and expanding the genetic basis of resisting the gibberellic disease of the wheat and is one of effective ways for improving the resistance of the wheat to the gibberellic disease.

Thinopyrum elongatum (Thinopyrum elongatum) is an important closely related species of wheat, and contains a plurality of genes which are extremely useful for improving wheat, such as disease resistance, cold resistance, drought resistance, salt and alkali resistance, aphid resistance and the like, wherein 1E and 7E chromosomes carry gibberellic disease resistance genes (see the reference: Liu-Dan, Zheng Liang, Wang Shi, Hou Yong, Lanxiu jin, Wei Youmiming, Lophopyrum elongatum chromosome location which influences the gibberellic disease resistance of wheat, university of Sichuan university, 2001,19: 200-.

Through years of research, a common wheat-diploid elytrigia elongata substitution line is hybridized with common wheat, and a wheat-elytrigia elongata 7EL brachial integral translocation line (T7BS.7EL) is obtained by an ionizing radiation method, and is identified to have excellent gibberellic disease resistance (see references: xylonite, Chenopodium, Lihaofeng, Liuhui duckweed, Dayi, heroic, Chenjian. wheat-elytrigia elongata 7E gibberellic disease resistance translocation line cultivation. Chinese agricultural science 2016, 49 (18): 3477-. However, the materials are all of the whole-arm translocation line, the agronomic characters of the materials are not outstanding, and the materials are not directly applied to breeding. In order to further transfer and utilize scab-resistant genes on a long-spike elytrigia repens 7EL chromosome, T7BS.7EL pollen after ionizing radiation is awarded to Yangmai 158, multiple backcrosses are carried out on the Yangmai 158, 7EL chromosome molecular markers and genome fluorescence in situ hybridization (GISH) identification are utilized in a radiation backcross progeny, and a large number of wheat-long-spike elytrigia repens 7EL small fragment translocation lines are created. Through the identification of the resistance to gibberellic disease for many years, the translocation line SF7EL9 is found to have excellent resistance to gibberellic disease, and a new germplasm is provided for wheat scab resistance breeding. The invention discloses a breeding method for improving the resistance of wheat scab by using the translocation line on the basis.

Disclosure of Invention

The invention aims to solve the problems, and provides a breeding method for improving the scab resistance of wheat by utilizing a scab resistant germplasm SF7EL9, namely, taking SF7EL9 as one of parents, identifying a translocation chromosome by utilizing a elytrigia elongata 7EL chromosome specific molecular marker Th7EL-46 and genome fluorescence in situ hybridization (GISH) on the basis of rolling backcross, and obtaining a new scab resistant wheat germplasm through a scab resistance test. Application of the gibberellic disease resistant germplasm SF7EL9 in molecular breeding for improving wheat scab resistance.

The purpose of the invention is realized by the following technical scheme: a breeding method for improving wheat scab resistance by using a scab resistant germplasm SF7EL9 is characterized by comprising the following steps:

step 1), breeding with the gibberellic disease resistant germplasm SF7EL9 as parent, namely male parent or female parentHybridization of a variety with good middle-high yield, F₁Backcrossing the generation or hybridizing with another variety with better yield to obtain BC₁F₁100 seeds;

step 2), planting BC by single plant₁F₁In seedling stage, molecular marker Th7EL-46 is used to pair BC₁F₁Selecting plants, reserving plants containing the Th7EL-46 marker, and harvesting seeds from the plants to obtain BC₁F₂Seeds;

step 3), planting BC by single plant₁F₂Selecting by using molecular marker Th7EL-46 and genome fluorescence in situ hybridization GISH at seedling stage, and obtaining seeds of plants containing Th7EL-46 marker and translocation chromosome by dividing the plants to obtain BC₁F₃Seeds;

step 4), planting BC by single plant₁F₃Line, every 10 BC₁F₃The strain is planted with a contrast parent 1 and a parent 2, wherein the parent 1 is SF7EL9 or Yangmai 23 or Annong 8455, and the parent 2 is Yangmai 23 or SF7EL9 Annong 8455; selecting plants containing Th7EL-46 marker and translocation chromosome in seedling stage, selecting plants with plant height, plant type and gibberellic disease resistance superior to those of control parent in grain filling stage and yellow falling stage, and harvesting seeds to obtain BC₁F₄Seeds;

step 5), planting BC by single plant₁F₄Line, every 10 BC₁F₄The strain is planted with a contrast parent 1 and a parent 2, wherein the parent 1 is SF7EL9 or Yangmai 23 or Annong 8455, and the parent 2 is Yangmai 23 or SF7EL9 Annong 8455; selecting plants containing Th7EL-46 marker and a pair of translocation chromosomes in seedling stage, selecting plant lines with plant height, plant type and gibberellic disease resistance superior to those of control parents in grain filling stage and yellow falling stage, and harvesting seeds by single plant to obtain BC₁F₅Seeds;

step 6), planting BC by single plant₁F₅Line, every 10 BC₁F₅The strain is planted with a contrast parent 1 and a parent 2, wherein the parent 1 is SF7EL9 or Yangmai 23 or Annong 8455, and the parent 2 is Yangmai 23 or SF7EL9 Annong 8455; selecting the composition at seedling stageTh7EL-46 marker and a pair of translocated chromosome plants, selecting plant lines with plant height, plant type and gibberellic disease resistance superior to those of a control parent in grain filling stage and yellow falling stage, and harvesting seeds in a mixed manner to obtain BC₁F₆Seeds;

step 7), planting BC in different districts₁F₆Seeds and three cells, and simultaneously planting a control parent 1 and a parent 2, wherein the parent 1 is SF7EL9 or Yangmai 23 or Annong 8455, and the parent 2 is Yangmai 23 or SF7EL9 Annong 8455; identification of gibberellic disease resistance at flowering time, Mixed harvest of BC₁F₆And (3) drying the seeds, weighing, and keeping the yield to be obviously higher than that of the control parent 1 and the parent 2, so as to obtain the new scab resistant wheat strain with high yield.

1 specific marker Th7EL-46 for tracing thinopyrum elongatum 7EL chromosome segment in a new scab-resistant wheat strain, wherein the molecular marker primer is Th7 EL-46F: TTACACTAACCCATGGTGTT, Th7 EL-46R: GCAGAGAATGAAGCAAAATC, the primer is used to perform PCR amplification on the filial generation line, a specific band of 7EL 450bp can be amplified, and the marker can effectively trace the 7EL translocation chromosome.

The method is advanced and scientific, and through the method, the breeding method for improving the scab resistance of the wheat by utilizing the scab resistant germplasm SF7EL9 takes SF7EL9 as a parent (a male parent or a female parent) to be hybridized with a variety with better high yield in breeding, F₁Backcrossing the generation or hybridizing with another variety with better yield to obtain BC₁F₁About 100 seeds are planted in a single plant BC₁F₁In seedling stage, molecular marker Th7EL-46 is used to pair BC₁F₁Selecting plants, reserving the plants containing the Th7EL-46 marker, and harvesting seeds of the individual plants; planting BC in single plant₁F₂Selecting by using molecular marker Th7EL-46 and genome fluorescent in situ hybridization (GISH) in seedling stage, and harvesting seeds by individual plants to obtain BC₁F₃Seeds; planting BC in single plant₁F₃Line, every 10 BC₁F₃Plant line planting is carried out by respectively selecting plant height, plant type and gibberellic disease resistance in grain filling stage and yellow falling stage according to the control parents 1 and 2Selecting plants containing Th7EL-46 marker and translocation chromosome from the lines of the control parents 1 and 2, and harvesting seeds of the plants individually to obtain BC₁F₄Seeds; planting BC in single plant₁F₄Line, every 10 BC₁F₄Planting the control parents 1 and 2 in the strain, selecting the strains with plant height, plant type and gibberellic disease resistance superior to those of the control parents 1 and 2 in the grain filling stage and the yellow falling stage, selecting the plants containing Th7EL-46 marker and a pair of translocation chromosomes, and harvesting the grains by separating the plants to obtain BC₁F₅Seeds; planting BC in single plant₁F₅Line, every 10 BC₁F₅The plants were grown as control parents 1 and 2, and BC was taken before sowing₁F₅Plant root tips are identified by genome fluorescence in situ hybridization (GISH), strains containing a pair of elytrigia elongata 7EL translocation chromosome segments are reserved, seeds are harvested in a mixed manner, and BC is obtained₁F₆Seeds; planting BC in subdistrict₁F₆Seeds, triplicate, and simultaneous planting of control parents 1 and 2, mixed harvest of BC₁F₆And (3) drying the seeds in the sun, weighing, and keeping the yield to be obviously higher than that of the 1 and 2 families of the control parent, so as to obtain the new scab-resistant wheat strain with high yield.

1 specific marker Th7EL-46 for tracing the elytrigia elongata 7EL chromosome segment in the new scab-resistant wheat strain is a molecular marker primer which is a primer consisting of Th7 EL-46F: TTACACTAACCCATGGTGTT, Th7 EL-46R: GCAGAGAATGAAGCAAAATC, the primer is used to perform PCR amplification on the filial generation line, so that a specific band of about 450bp of 7EL can be amplified, and the marker can effectively trace the 7EL translocation chromosome. In addition, fluorescent in situ hybridization (GISH and FISH) identification can effectively distinguish whether the translocation chromosome is homozygous or heterozygous, thereby facilitating molecular marker-assisted selective breeding.

The invention has the beneficial effects that: the common wheat-elytrigia elongata 7EL translocation line SF7EL9 is a gibberellic disease resistant small-fragment translocation line, is stable in heredity, carries elytrigia elongata 7EL chromosome fragments, has outstanding gibberellic disease resistance (figure 1), can obviously improve the gibberellic disease resistance of common wheat when being used for molecular breeding, and is convenient for culturing a new gibberellic disease resistant wheat strain.

The 1 Th7EL specific molecular marker Th7EL-46 disclosed by the invention can effectively identify whether the thinopyrum elongatum 7EL chromosome fragment is contained, thereby facilitating molecular marker-assisted selective breeding. After gel electrophoresis of the product amplified by the marker, the 450bp band was a 7EL chromosome fragment-specific band (FIG. 2). In addition, the identification combined with fluorescence in situ hybridization (GISH and FISH) can effectively distinguish whether the translocation chromosome is homozygous or heterozygous, and if only 1 translocation chromosome is contained, the material is a heterozygous translocation line; if 2 translocation chromosomes with the same FISH karyotype are contained, the material is shown to be a homozygous translocation line (FIG. 3).

In conclusion, the invention discloses a breeding method for improving the resistance of wheat scab. Mainly utilizes a distant hybridization means to hybridize the scab resistant wheat-elytrigia elongata 7EL small fragment translocation line SF7EL9 with cultivated wheat, and breeds the scab resistant wheat germplasm in the hybridization offspring. The Th7E-46 provided by the invention can be used as a biomarker to be applied to real-time identification of the elytrigia elongata 7EL chromosome segment contained in disease-resistant germplasm.

Drawings

FIG. 1 shows the field phenotype and gibberellic disease resistance of the small fragment translocation line SF7EL 9;

a: phenotype of small fragment translocation line SF7EL9 following field heading; b: single-flower inoculation of scytha graminearum on the basis of 21-day after-ear phenotype was performed on small-fragment translocation line SF7EL9, susceptible variety Annong 8455(AN8455), Roblin, and disease-resistant variety Sumai No. 3 (SU 3).

FIG. 2 shows the PCR amplification result of the specific molecular marker Th7EL-46 in the small fragment translocation line SF7EL 9; m: DNA Marker; 1: the 7EL small fragment metathesis system SF7EL 9; 2: 7EL Whole-arm metathesis system T7 BS.7EL; 3: the attachment system DA 7E; 4: 2X of diploid thinopyrum elongatum; 5: common wheat, Chinese Spring (CS); 6: wheat, triticum aestivum 158.

FIG. 3 shows the results of in situ hybridization (GISH) of mitotic metaphase chromosomal genomes of small fragment translocation lines SF7EL9 root tip cells; the green fluorescence signal is the thinopyrum elongatum 7EL chromosome fragment, and the wheat chromosome is red.

FIG. 4 shows the results of PCR and GISH identification of the backcross progeny of SF7EL9 and Annon 8455;

a, PCR amplification results of a specific molecular marker Th7EL-46 in filial backcross progeny. M: DNA Marker; 1: the 7EL small fragment metathesis system SF7EL 9; 2: 7EL Whole-arm metathesis system T7 BS.7EL; 3: filial backcross progeny; 4: 2X of diploid thinopyrum elongatum; 5: common wheat, Chinese Spring (CS); 6: common wheat Annong 8455.b, in-situ hybridization (GISH) of root tip cell mitosis metaphase chromosome genome of a hybridization backcross progeny. In the figure, a red arrow emits a green fluorescence signal, the red arrow is a thinopyrum elongatum 7EL chromosome fragment, and the wheat chromosome is red.

FIG. 5 shows the resistance of gibberellic disease of the progeny of the backcross of small-fragment translocation line SF7EL9 and Annong 8455;

the ear phenotype of the small fragment translocation line SF7EL9, susceptible variety Annong 8455(AN8455), Roblin cross backcross progeny (xAN 8455), and disease-resistant variety Sumai No. 3 (SU3) after 21 days by single flower instillation of fusarium graminearum.

FIG. 6 shows PCR identification and gibberellic disease resistance of backcross progeny of small fragment translocation line SF7EL9 and Yangmai 23;

a, PCR amplification results of a specific molecular marker Th7EL-46 in filial backcross progeny; m: DNA Marker; 1: the 7EL small fragment metathesis system SF7EL 9; 2: 7EL Whole-arm metathesis system T7 BS.7EL; 3: filial backcross progeny; 4: 2X of diploid thinopyrum elongatum; 5: common wheat, Chinese Spring (CS); 6: common wheat Yangmai 23.b, small fragment translocation line SF7EL9, susceptible variety Annong 8455(AN8455), Roblin, parent Yangmai 23(Y23), filial backcross progeny (YZU-7EL049) and disease-resistant variety Sumai No. 3 (SU3) are inoculated with fusarium graminearum in a single flower mode for 21 days to obtain ear phenotype.

Detailed Description

The present invention will now be described in further detail with reference to the following examples, figures and accompanying illustrations, which are intended to be illustrative, but not limiting, of the invention.

Example 1

In order to understand the effect of a small-fragment translocation line SF7EL9 on scab resistance and to clarify the utilization value of the line, SF7EL9 is used as a male parent, an susceptible variety Annong 8455 is used as a female parent for hybridization, then the Annong 8455 is used as the female parent for backcross for 1 generation and then selfing is carried out, and a molecular marker Th7EL-46 and genome fluorescence in situ hybridization (GISH) are used for identification (figure 4), so that improved new germplasm is obtained. The resistance identification of the gibberellic disease for many years shows that the obtained new germplasm is highly resistant to the gibberellic disease (figure 5).

Example 2

Using SF7EL9 as one of parents (male parent or female parent) to hybridize with the high-yield variety Yangmai 23. First, SF7EL9 was hybridized with Yangmai 23, F₁Backcrossing the generation with Yangmai 23 to obtain BC₁F₁Seed, Individual planting BC₁F₁Selecting by using a molecular marker Th7EL-46 at the seedling stage, reserving plants containing the Th7EL-46 marker, and harvesting the plants individually to obtain BC₁F₂Seeds; planting BC in single plant₁F₂Selecting each single plant by using molecular markers and GISH, reserving the single plant containing a Th7EL-46 marker plant and a thinopyrum elongatum chromosome, simultaneously carrying out gibberellic disease resistance identification by using a single-flower drip method in the flowering period, and harvesting the plants with good gibberellic disease resistance to obtain BC by using the single plant₁F₃Seeds; planting BC in single plant₁F₃Selecting each single plant by using molecular markers and GISH, reserving plants containing Th7EL-46 markers and a pair of translocation chromosomes, performing gibberellic disease resistance identification by using a single flower drip method in the flowering period, performing agronomic character statistics, and harvesting plants with good gibberellic disease resistance and agronomic characters from the single plants to obtain BC (plant block C)₁F₄Seeds; planting BC in single plant₁F₄Selecting each individual plant by using molecular markers and GISH, reserving plants containing Th7EL-46 marker plants and a pair of translocation chromosomes, performing gibberellic disease resistance identification by using a single flower drip method in the flowering period, performing agronomic character statistics, and harvesting plants with good gibberellic disease resistance and agronomic characters in a mixed mode to obtain BC (plant block c) by using each individual plant to breed 6 rows and 5 plants in each row₁F₅Seeds; planting BC in subdistrict₁F₅And 23, 3 times of repetition of the parent Yangmai, and counting the agronomic characters to finally obtain the new gibberellic disease resistant germplasm YZU-7EL049 (table 1, figure 6).

TABLE 1 comparison of the novel germplasm YZU-7EL049 with its background parent Yangmai 23 for major agronomic traits

Note: different letters in the table indicate a significant level of difference of 0.05.

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