JP6876334B2 - How to make transformation-sensitive barley - Google Patents

Description

The present invention relates to a genomic region from the barley variety Golden Promise that is involved in susceptibility to transformation by the Agrobacterium method. The present invention also relates to a method for producing barley sensitive to transformation by the Agrobacterium method, using the presence of the genomic region as an index. Furthermore, the present invention relates to reagents for testing susceptibility to transformation by the Agrobacterium method in barley, which comprises an oligonucleotide that binds to the genomic region.

Cultivated barley (Hordeum vulgare) is a diploid grain with a well-characterized genome of 5.1 Gbps. Barley ranks fourth in the world's production of grains and is used in food, brewing, and animal feed. Due to its importance as a crop, many agricultural or industrial characteristics of barley have been genetically analyzed. Transformation is an essential tool in genetics, and the introduction of DNA into the biological genome enables not only the introduction of new traits but also the analysis of endogenous genes. Unfortunately, genetic analysis in barley is constrained by technical problems with transformation, which is in stark contrast to the ease of transformation by the Agrobacterium method in other plant species such as Arabidopsis and rice. is there. The most widely used transformation method by the Agrobacterium method in barley was developed by Tingay et al. (Non-Patent Document 1), and immature embryos of Golden Promise are used as explants. As another barley transformation technique, pollen culture of Igri is one of the few alternative methods (Non-Patent Document 2). This situation poses a great challenge to complementary analysis in barley, as the analysis of the traits of interest is limited to alleles expressed in golden promises and iguri. In general, functional allele analysis requires the development of strains in which other genomic regions are replaced with the golden promise type, while retaining the target allele through multiple backcrosses with the golden promise. Recently, Yeo et al. (Non-Patent Document 3) have inherited the transforming ability of Golden Promise and rust inherited from SusPtrit in order to develop a barley strain suitable for functional analysis of non-hostability and partial resistance to rust fungi. We have developed a new barley strain called Golden SusPtrit, which is highly susceptible to disease. The enhancement of transformation efficiency by the Agrobacterium method in this recombinant line suggested the existence of genetic factors that are the key to the transformation efficiency of Golden Promise. In this study, transformation-sensitive and insensitive strains (SG062N) and insensitive strains (SG133N) in the same haploid mapping population were used to predict the genetic regions responsible for Agrobacterium transformation in the golden promise. We also compared the genetic differences between them (Non-Patent Document 3).

Although the genetic factors of transformation ability itself have not been specifically investigated, the four quantitative trait loci (QTLs) that are key to the regeneration of callus to green shoots in Molex have previously been Steptoe. Was identified in a hybrid between Molex and Morex (Non-Patent Documents 4 and 5). In Chinese cabbage (Brassica oleracea), Cogan et al. (Non-Patent Document 6) identified three QTLs involved in root transformation with Agrobacterium lysogenes using ripe seeds. Two of the three loci are presumed to be paralogs of each other resulting from genomic duplication around these regions (Non-Patent Document 7). In potatoes (Solanum tuberosum), El-Kharbotly et al. (Non-Patent Document 8) found a locus involved in transformation efficiency by linking with the R1 gene (resistance to the Phytophthora infestans). In the case of rice, Nishimura et al. (Non-Patent Document 9) isolated a gene encoding ferredoxin-nitrate reductase (NiR), which is involved in the promoter of shoot regeneration (PSR), which is a major QTL. did. In this case, the regenerative efficiency of rice in Koshihikari was increased by introducing the complete genomic region or overexpressing cDNA of the NiR gene of Casalas into callus derived from ripe seeds. Interestingly, Tyagi et al. (Non-Patent Document 10) reported that the QTL on barley chromosome 6H, which is the key to green shoot regeneration, is located near the locus (AK371794).

The efficiency of transformation by the Agrobacterium method largely depends not only on the type and genetic composition of the plant material being investigated, but also on environmental and technical factors. Technical factors include culture conditions, medium composition, Agrobacterium strain type, and specific binary vectors and selectable markers to employ (Non-Patent Document 11). For transformation as well as genetic factors involved in interaction with Agrobacterium, including genes that are key to T-DNA integration into the host genome, cell division under selective conditions, and regeneration from callus. The type of explant used also plays an important role (Non-Patent Document 11).

Recently, genome editing technologies such as TALENs (Trans Activator-Like Effector Nucleases) and CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated proteins 9) have been developed in plants. However, the practicality of these techniques in barley was the first to introduce these nucleases in most plant species, as reported by Budhagatapalli et al. (Non-Patent Document 12) and Lawrenson et al. (Non-Patent Document 13). Constrained by the fact that transformation is required as a step.

Tingay, S. et al., (1997) Plant J., 11, 1369-1376. Kumlehn, J. et al., (2006) Plant Biotechnol. J., 4, 251-261. Yeo, F.K.S. et al., (2014) Theor. Appl. Genet., 127, 325-337. Mano, Y. et al., (1996) Japanese Journal of Breeding, 46, 137-142. Bregitzer, P. and Campbell, R.D. (2001) Crop Sci., 41, 173-179. Cogan, N. et al., (2002) Theor. Appl. Genet., 105, 568-576. Cogan, N.O.I. et al., (2004) Plant Biotechnol. J., 2, 59-69. El-Kharbotly, A. et al., (1995) Theor. Appl. Genet., 91, 557-562. Nishimura, A. et al., (2005) Proc. Natl. Acad. Sci. USA, 102, 11940-11944. Tyagi, N. et al., (2010) Crop Sci., 50, 1697-1707. Cheng, M. et al., (2004) In Vitro Cell Dev. Biol. Plant, 40, 31-45. Budhagatapalli, N. et al., (2015) G3: Genes Genom. Genet., 5, 1857-1863. Lawrenson, T. et al., (2015) Genome Biol., 16, 1-13.

The present invention has been made in view of such a situation, and an object of the present invention is to identify a genomic region involved in the efficiency of transformation of barley by the Agrobacterium method, and to use the existence of the genomic region as an index. The object of the present invention is to provide a method for producing barley that is sensitive to transformation by the Agrobacterium method.

Successful transformation in plants depends on many factors, and immature embryos of Golden Promise (GP) are the most reliable material for Agrobacterium transformation in barley (Harwood, WA (2012). ) J. Exp. Bot., 63, 1791-1798.). Therefore, the present inventor first attempted a transformation experiment using a BC3F8 recombinant chromosomal substitution line in order to identify a genetic factor that controls transformation by the Agrobacterium method in barley. However, transformed plants could not be obtained from 4,661 immature embryos. It is presumed that the number of factors required for transformation in the experimental material was insufficient.

Therefore, we have adopted a different approach to increase the chances of creating a transformant population for further study. That is, as a material for transformation by the Agrobacterium method, F2 generation immature embryos obtained from the cross between Haruna Nijo (HN) and GP were used. This new approach succeeded in obtaining 60 transformed barley plants (HN x GP) from 3030 F2 individuals.

For genome-wide analysis, these 60 transformed plants were then used to attempt to identify genomic regions showing significant allele segregation distortion from the expected 1: 2: 1 ratio. As a result, the inventor found the major regions that cause segregation distortion on chromosomes 2H and 3H and named them TFA1, TFA2, and TFA3. There, the GP allele frequency was very high in the transformants. A certain amount of TFAs heterozygous individuals were also present in the transformed plants, suggesting that some potential factor controlling the transformation efficiency is dominant. Importantly, these TFAs GP alleles were also conserved in two transformed plants from the alternative cross MO × GP.

Thus, the present inventor has succeeded in identifying for the first time in the world the genomic region underlying the transformation susceptibility of barley by the Agrobacterium method by measuring the segregation strain of alleles in the F2 population. Then, by using the presence of the genomic region as an index, it is possible to efficiently produce transformation-sensitive barley by the Agrobacterium method, and the oligonucleotide that binds to the genomic region is Agrobacterium. We have found that it is useful for testing barley that is susceptible to transformation by the method, and have completed the present invention.

The present invention provides, in more detail,:

[1] The golden promise is crossed with any barley variety, and in the obtained progeny, the presence of at least one genomic region (a) to (c) below is detected, and the genomic region is homozygous or heterozygous. A method for producing barley that is sensitive to transformation by the Agrobacterium method, including selection of individuals of the type.

(a) Genome region sandwiched between the marker NIASHv1109O03_00000798_3H and the marker NIASHv1150F05_00000946_3H on chromosome 3H
(b) Genome region sandwiched between the marker NIASHv2027F11_00000300_2H and the marker NIASHv2016D11_00000337_2H on chromosome 2H
(c) Genome region sandwiched between the marker NIASHv2108A23_00000279_2H and the marker FLOUbaf102a14_00001505_2H on chromosome 2H [2] At least one marker of the following (a) to (c) in the progeny obtained by crossing the golden promise with any worm variety. A method for producing a corn that is sensitive to transformation by the Agrobacterium method, which comprises detecting the presence of the marker and selecting an individual whose marker is homozygous or heterozygous and has a golden promise type.

(a) A marker selected from the group consisting of the marker NIASHv1109O03_00000798_3H on chromosome 3H, the marker NIASHv1150F05_00000946_3H, and the markers existing in the genomic region sandwiched between these markers.
(b) A marker selected from the group consisting of the marker NIASHv2027F11_00000300_2H on chromosome 2H, the marker NIASHv2016D11_00000337_2H, and the markers existing in the genomic region sandwiched between these markers.
(c) A marker selected from the group consisting of the marker NIASHv2108A23_00000279_2H on chromosome 2H, the marker FLOUbaf102a14_00001505_2H, and the markers existing in the genomic region sandwiched between these markers. A reagent for testing susceptibility to transformation by the Agrobacterium method in caterpillars, which comprises a pair of oligonucleotides designed to be sandwiched.

(a) A marker selected from the group consisting of the marker NIASHv1109O03_00000798_3H on chromosome 3H, the marker NIASHv1150F05_00000946_3H, and the markers existing in the genomic region sandwiched between these markers.
(b) A marker selected from the group consisting of the marker NIASHv2027F11_00000300_2H on chromosome 2H, the marker NIASHv2016D11_00000337_2H, and the markers existing in the genomic region sandwiched between these markers.
(c) A marker selected from the group consisting of the marker NIASHv2108A23_00000279_2H on chromosome 2H, the marker FLOUbaf102a14_00001505_2H, and the markers existing in the genomic region sandwiched between these markers [4] At least one marker of (a) to (c) below. A reagent for testing the susceptibility of a wheat to transformation by the Agrobacterium method, which comprises an oligonucleotide that hybridizes to a base sequence on the containing genome.

(a) A marker selected from the group consisting of the marker NIASHv1109O03_00000798_3H on chromosome 3H, the marker NIASHv1150F05_00000946_3H, and the markers existing in the genomic region sandwiched between these markers.
(b) A marker selected from the group consisting of the marker NIASHv2027F11_00000300_2H on chromosome 2H, the marker NIASHv2016D11_00000337_2H, and the markers existing in the genomic region sandwiched between these markers.
(c) A marker selected from the group consisting of the marker NIASHv2108A23_00000279_2H on chromosome 2H, the marker FLOUbaf102a14_00001505_2H, and the markers existing in the genomic region sandwiched between these markers.

By selecting individual barley using the genomic region found in the present invention as an index, it has become possible to increase the transformation efficiency of barley by the Agrobacterium method. According to the present invention, it has become possible to efficiently produce transformants even in barley varieties that have been difficult to transform.

It is an overall drawing of the genotype of a transformed HN × GP plant. Genotyping of transformed HN × GP plants Nos. 1 to 60 (from left to right in the figure) was performed using the 384-SNP platform of the Illumina GoldenGate (R) assay. 124 SNP markers were shown according to the marker position (MP) on each chromosome based on barley genomic information (IBGSC 2012). Alleles from Haruna Nijo (HN), heteroregions (HE), and alleles from Golden Promise (GP) are shown in light gray, dark gray, and black, respectively. The numerical value (cM) shown on the left indicates the marker position at the end of each chromosome. It is a graph which separated and analyzed the allele and SNP marker in the transformed HN × GP plant. Allelic isolation (A) and chi-square (χ2) values (B-C) were calculated by genotyping of 124 SNP markers in transformed HN × GP plants. SNP markers were shown from short arm (left) to long arm (right) on each chromosome according to the order and genetic distance of the markers in barley genomic information (IBGSC 2012). The numerical value (cM) shown at the bottom of the figure indicates the marker position at the end of each chromosome. (A) Shows the percentage of golden promise (GP), Haruna Nijo (HN), and hetero (HE) alleles for each SNP marker. (B) The polygonal line is the χ2 value calculated using 1: 2: 1 as the expected separation ratio. Dotted lines show statistically significant differences at the 1% and 5% levels (df = 2). (C) The polygonal line shows the χ2 value calculated using 3: 1 as the expected separation ratio for [HN + HE]: [GP] and [GP + HE]: [HN]. The two dotted lines show statistically significant differences at the 1% and 5% levels, respectively (df = 1). TFAs (TFA1-10) are loci that exhibit segregation distortion. It is a figure and electrophoretic photograph which show the genetic structure and allele of the HvNiR gene in the transformed HN × GP plant. (A) The structure of the HvNiR gene region containing an intron, a start codon, and a stop codon is shown. White and gray wedges indicate single nucleotide polymorphisms (SNPs) and insertion / deletion of two or more bases between Haruna Nijo and Golden Promise, respectively. The insertion of a 161 bp retrotransposon-like sequence was present in the first intron of HvNiR in the golden promise. (B) Genotyping was performed on transformed HN × GP plants using HvNiR-specific primers. Expected sizes of PCR fragments were 280 bp and 417 bp in the Haruna Nijo (H) and Golden Promise (G) alleles, respectively. M indicates a ladder marker of 100 bp, and 1-60 indicates an individual transformed HN × GP plant. It is a figure which shows the result of the analysis of the locus-locus interaction in the transformed HN × GP plant. (A) Among transformed HN × GP plants with alleles of HvNiR GP (top), HE (center), and HN (bottom), golden promise (GP, black), hetero (HE, dark gray), and The ratio of alleles of Haruna Nijo (HN, light gray) was calculated and shown as a band chart. 110 SNP markers and HvNiR gene markers were shown from the short arm (left) to the long arm (right) on the chromosome. TFAs (TFA1-10) are loci showing segregation distortion in FIG. A color-coded diagram based on the marker chi-square (χ2) test (df = 2) is shown below each band graph. It is a figure which shows the result of the analysis of the locus-locus interaction in the transformed HN × GP plant. (B) A heat map was created based on the p-value of the χ2 test (df = 2), which represents the significance of segregation strain in transformed HN × GP plants. The marker (locus A) is grouped into GP (left panel), HE (center panel), and HN (right panel) alleles, and each chromosome is on the vertical axis from short arm (bottom) to long arm (top). Organized in. Then, a χ2 test (df = 2) was performed on all other markers (locus B) arranged on the horizontal axis from the short arm (bottom) to the long arm (top) on each chromosome. The p-value, which indicates the degree of significance, is indicated by the degree of color striking. It is a graph which shows the frequency of the transformed HN × GP plant classified by the allele type in TFA1, TFA2 and TFA3. It is an overall view of the universal complementary system of barley based on the proposed TFA. Due to the complementarity test of the target gene, F2 plants can be produced by crossing between the target variety having the target gene and the golden promise. Genotyping can be performed among F2 plants to identify plants with GP alleles in TFA1 and TFA2, and GP or hetero in TFA3, as well as homozygotes for the target gene. It is estimated that plants that serve this purpose are found in one proportion among approximately 86 F2 plants.

<Method of producing barley sensitive to transformation by Agrobacterium method>
The present invention provides a method for producing barley that is sensitive to transformation by the Agrobacterium method.

In the present invention, "transformation by the Agrobacterium method" means a method of transforming a plant by introducing a foreign gene into the genome of a plant cell via Agrobacterium. The Agrobacterium used is not particularly limited, and examples thereof include Agrobacterium tumefaciens, Agrobacterium bitis, Agrobacterium rhizogenes, and Agrobacterium radiobacter. As a specific method, for example, the binary vector method can be preferably used.

In the method of the present invention, first, a golden promise is crossed with an arbitrary barley variety. Golden promise has been suitably used as a material for transformation by the Agrobacterium method in barley, but the present inventor has a genomic region considered to contain a genetic factor that controls the transformation. Succeeded in identifying. Therefore, the golden promise is crossed with any barley variety, the presence of the genomic region derived from the golden promise is detected in the progeny, and if the genomic region is present, the progeny is transformed by the Agrobacterium method. On the other hand, it can be evaluated that it is highly probable that it is a sensitive barley.

The barley varieties to be crossed with Golden Promise are not particularly limited as long as they can be crossed. A desired barley variety can be used that wants to impart or increase susceptibility to transformation by the Agrobacterium method. Examples of barley varieties include, but are not limited to, Nijo barley such as Haruna Nijo.

The progeny is not particularly limited, but is preferably an F2 plant from the viewpoint of efficiently producing barley sensitive to transformation by the Agrobacterium method.

In the method of the present invention, the presence of at least one genomic region (a) to (c) below is then detected in the obtained progeny.

(a) Genome region sandwiched between the marker NIASHv1109O03_00000798_3H and the marker NIASHv1150F05_00000946_3H on chromosome 3H (hereinafter referred to as the "first target genomic region").
(b) Genome region sandwiched between the marker NIASHv2027F11_00000300_2H and the marker NIASHv2016D11_00000337_2H on chromosome 2H (hereinafter referred to as "second target genomic region").
(c) A genomic region sandwiched between the marker NIASHv2108A23_00000279_2H and the marker FLOUbaf102a14_00001505_2H on chromosome 2H (hereinafter referred to as the "third target genomic region").
The first target genomic region contains TFA1, the second target genomic region contains TFA2, and the third target genomic region contains TFA3.

In the present invention, the presence of at least one of the above three target genomic regions is targeted for detection, but preferably two, and most preferably all three are targeted for detection. Further, it is not necessary to detect the entire target genomic region, and a part thereof may be detected. In fact, in each target genome region, if even a part of it is a golden promise type, it is highly probable that the whole is a golden promise type (Tables 1 to 9 below), so even if it is a part of the detection. , Useful in the selection of barley sensitive to transformation by the Agrobacterium method.

In detecting the presence of each target genomic region, a DNA sample is first prepared from the test barley. As the barley for extracting DNA, not only grown plants but also seeds and seedlings can be used. As the tissue for extracting the DNA sample, for example, leaves can be used. The method for extracting genomic DNA from barley is not particularly limited, and a known method can be appropriately selected and used, and examples thereof include an SDS phenol method, a CTAB method, and an alkali treatment method. Commercially available kits such as the DNeasy Plant mini kit (QIAGEN, Germany) can also be used.

Markers can be used to determine whether the target genomic region is of the golden promise type. In the present invention, the "marker" means a specific genomic region on a chromosome capable of distinguishing a golden promise from other barley varieties.

Tables 1-9 show the SNP markers found between Golden Promise and Haruna Nijo.

In addition, the nucleotide sequences of the peripheral genomic regions containing each SNP marker are shown in SEQ ID NOs: 1-190 (these are double-stranded DNAs, but they are shaded in the "SEQ ID NO:" column in the table. In the SEQ ID NO:, the antisense strand is described in the sequence listing. On the other hand, in the unshaded SEQ ID NO:, the sense strand is described in the sequence listing). In the present invention, the presence of the above-mentioned genomic region can be detected by using the fact that these SNP markers are golden promise type bases as an index (bases of each SNP on the sense chain side and golden promise type bases [double]. Body genome] is shown in the table).

Detection of SNPs in the target genomic region can be performed by isolating the DNA containing the SNP site and determining the base sequence of the isolated DNA. Isolation of the DNA can be performed, for example, by PCR using a pair of oligonucleotides designed to sandwich the SNP site and using genomic DNA as a template. The base sequence of the isolated DNA can be determined by a method known to those skilled in the art, such as the Sanger method and the Makisam Gilbert method.

Another method for detecting SNPs is the PCR-SSP (PCR-sequence-specific primer) method. In this method, the 3'end of one of the pair of oligonucleotides constituting the primer is designed to be a base species complementary to a specific base species at the SNP site. PCR using a pair of oligonucleotides designed in this way is amplified only when genomic DNA derived from a corn variety having a specific base species of SNP site is used as a template, and the SNP site is different. When genomic DNA derived from a varieties of Omugi, which is a base species, is used as a template, it is not amplified. SNP can be detected using the presence or absence of such amplification as an index.

When restriction fragment length polymorphism (RFLP) can be set at the SNP marker site, for example, PCR-RFLP method (or CAPS [Cleaved Amplified Polymorphic Sequence] method) using those RFLP markers as an index. It can also be detected by such as.

These markers in the target genomic region can be appropriately set by those skilled in the art by comparing the base sequences of the respective target genomic regions in the golden promise and other barley varieties to be crossed. The nucleotide sequence of each barley variety is described, for example, in IPK Barley BLAST Server (http://webblast.ipk-gatersleben.de/barley/).

Yet another method for detecting SNP is the PCR-SSCP (PCR-single nucleotide polymorphism) method. Double-stranded DNA amplified by PCR using a pair of oligonucleotides designed to sandwich the SNP site is denatured by treatment with heat, alkali, etc. to make single-stranded DNA, and then a denaturing agent is used. When subjected to polyacrylamide gel electrophoresis without it, single-stranded DNA in the gel is folded by intramolecular interactions to form higher-order structures. Since the interaction of the folded structure changes depending on the difference in base type, the separated single-stranded DNA is detected by silver staining or radioisotope, and the mobility of the single-stranded DNA on the gel is used as an index. SNP can be detected.

Yet another method for detecting SNP is to use an intercalator. In this method, first, a DNA sample is prepared from the test barley. Then, in a reaction system containing an intercalator that fluoresces when inserted between DNA double strands, the region containing SNP is amplified using the DNA sample as a template. Then, the temperature of the reaction system is changed, the fluctuation of the fluorescence intensity emitted by the intercalator is detected, and the SNP can be detected by using the fluctuation of the fluorescence intensity accompanying the detected temperature change as an index. Examples of such a method include a high resolution melting curve analysis (HRM) method.

Yet another method for detecting SNPs is to utilize an oligonucleotide probe that hybridizes to a region containing SNPs. In one embodiment of this method, a DNA sample is first prepared from the test barley. On the other hand, oligonucleotide probes labeled with reporter fluorescent dyes and quencher fluorescent dyes are prepared by specifically hybridizing to the region containing SNP. Then, the oligonucleotide probe is hybridized with the DNA sample, and the DNA containing the SNP site is amplified using the DNA sample hybridized with the oligonucleotide probe as a template. Then, the fluorescence emitted by the reporter fluorescent dye whose suppression by the quencher is released is detected by the decomposition of the oligonucleotide probe accompanying the amplification. Examples of such a method include a double die probe method, a so-called TaqMan (registered trademark) probe method.

Other embodiments utilizing reporter fluorescent dyes and quencher fluorescent dye-labeled oligonucleotide probes include chimeric oligonucleotides (RNA-DNA chimera) and RNase H that specifically hybridize to regions containing SNP sites. It is also possible to utilize a cycling probe method that utilizes a combination with the enzyme of.

The detection of the marker in the present invention is not limited to the above embodiment. For example, modifier concentration gradient gel electrophoresis (DGGE method), invader method, pyrosequencing method, single nucleotide primer extension (SNuPE) method, allergen-specific oligonucleotide (ASO) hybridization method, ribonuclease A mismatch cleavage method, Other known techniques, such as the DNA array method, can also be utilized in the present invention.

In the detection of the SNP marker targeted in this example, for example, 384- of Illumina GoldenGate (R) oligonucleotide pool assays (Close, TJ et al., (2009) BMC Genomics, 10, 1-13.) You can also use the SNP platform.

In the method of the present invention, an individual whose target genomic region is homozygous or heterozygous and has a golden promise type is then selected. As is clear from FIG. 5, not only individuals whose target genomic region is homozygous and golden promise type, but also individuals whose target genomic region is homozygous and golden promise type are Agrobacterium compared to the case where they are homozygous and dichotomous. It is highly probable that it is sensitive to transformation by the method. In the present invention, preferably, two or more of the three target genomic regions are homozygous or heterozygous and golden promise type, and more preferably all three are homozygous or heterozygous and golden promise type. To select.

In the most preferred embodiment, individuals are selected in which the first target genomic region and the second target genomic region are homozygous and golden promise type, and the third target genomic region is homozygous or heterozygous and golden promise type (FIG. 6). ). As a result, individuals susceptible to transformation by the Agrobacterium method can be efficiently selected.

<Reagent for testing susceptibility to transformation by the Agrobacterium method>
The present invention provides reagents for testing the susceptibility of barley to transformation by the Agrobacterium method.

One embodiment of the reagents of the present invention comprises a pair of oligonucleotides designed to sandwich the marker. Such oligonucleotides can be used, for example, as primers to amplify the genomic region containing the marker. As used herein, "designed to be sandwiched" means that the oligonucleotide is designed so that the amplification product of the pair of oligonucleotides contains a marker site. Therefore, for example, when the oligonucleotide is used for detecting SNP, depending on the detection method, one of the oligonucleotides of the pair of oligonucleotides contains a base sequence complementary to the base sequence of SNP. May be good.

Another aspect of the reagent of the present invention comprises an oligonucleotide that hybridizes to a base sequence on the genome containing the marker. Such oligonucleotides can be used, for example, as probes to detect genomic regions containing the markers. The oligonucleotide preferably hybridizes specifically to the marker site under normal hybridization conditions, preferably stringent hybridization conditions. The oligonucleotide may be labeled with a fluorescent dye, isotope, biotin or the like as appropriate. As a labeling method, a method of labeling by phosphorylating the 5'end of the oligonucleotide with 32P using T4 polynucleotide kinase, and a method of labeling by phosphorylating the 5'end of the oligonucleotide with 32P, and using a DNA polymerase such as Klenow enzyme, and using a random hexamer oligonucleotide or the like as a primer. As an example, a method of incorporating a substrate base labeled with an isotope such as 32P, a fluorescent dye, or biotin (random prime method or the like) can be exemplified.

The chain length of the above oligonucleotides (primers and probes) is at least 15 bases. It is usually 15 to 100 bases, preferably 17 to 30 bases. The oligonucleotide can be produced, for example, by a commercially available oligonucleotide synthesizer. The oligonucleotide probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like. Further, the oligonucleotide does not have to be composed only of natural nucleotides (DNA or RNA), and a part or all of them may be composed of non-natural nucleotides. Examples of non-natural nucleotides include PNA (polyamide nucleic acid), LNA (registered trademark, locked nucleic acid), and ENA (registered trademark, 2'-O, 4'-C-Ethylene-bridged nucleic acid acids). Be done.

In the reagent of the present invention, in addition to the oligonucleotide which is the active ingredient, for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, preservative and the like are mixed as necessary. May be.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.

[Materials and methods]
(1) Plant material In the hybrid F1 barley plant (HN × GP or MO × GP), the seed parent is derived from Haruna Nijo (HN) or Molex (MO), and the pollen parent is derived from Golden Promise (GP). .. The plants were grown at 15 ° C / 13 ° C (day / night) under 16 hours of sunshine per 24-hour cycle. Approximately 14 days after pollination, the grains were harvested to isolate immature embryos.

(2) Transformation by Agrobacterium method Transformation of barley was carried out according to the protocol of Hensel et al. (Hensel, G. et al., (2008) J. Plant Physiol., 165, 71-82). The cut immature embryos were subjected to Hiei et al. (Hiei, Y. et al., (2006) Plant Cell Tiss. Org. Cult., 87, 233-243.) And Zheng et al. (Zheng, L. et al., (2011). ) According to Plant Cell Physiol., 52, 765-774.), Soaked in 100 mg / l acetricirigon solution at 43 ° C. for 4 minutes before inoculation with Agrobacterium. Agrobacterium carrying a binary vector pIG121-Hm (Hiei, Y. et al., (1994) Plant J., 6, 271-282.) Having a beta-glucuronidase (GUS) gene and a hygromycin phosphotransferase (HPT) gene. -Tumefaciens strain AGL1 was used for inoculation.

(3) DNA analysis by PCR Genomic DNA was extracted from the leaves of barley plants using the DN easy Plant mini kit (QIAGEN, Germany). PCR amplification was performed in the following program. 28 cycles of initial denaturation at 95 ° C, 2 minutes, "denaturation at 95 ° C, 30 seconds, annealing at 55 ° C (for GUS gene) or 60 ° C (for HPT gene), annealing at 72 ° C, 30 seconds" (72 ° C, with final elongation for 10 minutes). The 10 μl reaction mixture for PCR contains 50 ng of genomic DNA as a template, 1 × GoTaq (R) Green Master Mix (Promega, USA), and 0.5 μM of each specific primer below.

For the GUS gene, gus4 (5'-AACAGTTCCTGATTAACCACAAACC-3' / SEQ ID NO: 191) and gus5 (5'-GCCAGAAGTTCTTTTCAGTACC-3' / SEQ ID NO: 192), and for the HPR gene, hph1 (5'-GCTGGGGCGTCGGTTTCCACTATCGG). -3'/ SEQ ID NO: 193) and hph2 (5'-CGCATAACAGCGGTCATTGACTGGAGC-3' / SEQ ID NO: 194).

(4) Cloning of HvNiR gene
PCR amplification was performed with the following program to clone the HvNiR genes (Acc. No. LC097010 and LC097011 for GP and HN, respectively). Start denaturation at 98 ° C, 2 minutes, "denaturation at 98 ° C, 10 seconds, primer annealing at 55 ° C, 15 seconds, and extension at 72 ° C, 150 seconds" for 30 cycles (72 ° C, with final extension for 10 minutes) ). 30 μl of the reaction mixture for PCR includes 50 ng of Haruna Nijo or Golden Promise genomic DNA as a template, 1 × PrimeSTAR (R) Max DNA Polymerase (TAKARA, Japan), and 0.5 μM specific primer HvNiR-F1 (5). Includes'-AACCACAAGCAGCATCCATG-3'/ SEQ ID NO: 195) and HvNiR-R1 (5'-GAGATCATCAGGAGAAGGAG-3' / SEQ ID NO: 196). PCR products were cloned into pCRTM4-TOPO (R) (Invitrogen, USA) and sequenced using TOPO (R) TA Cloning (R) Kit for Sequencing (Invitrogen, USA) according to the manufacturer's protocol.

(5) Genotyping Illumina GoldenGate (R) javax pool assays (Close) for genotyping of all transformed plants produced using genomic DNA extracted with DN easy Plant mini kit (QIAGEN, Germany) according to the manufacturer's protocol. , TJ et al., (2009) BMC Genomics, 10, 1-13.) 384-SNP platform was adopted.

Genotyping with HvNiR gene markers was performed using a touchdown PCR program with improved PCR amplification (Don, R.H. et al., (1991) Nucleic Acids Res., 19, 4008.). Start denaturation at 95 ° C, 2 minutes, "Melting at 95 ° C, 30 seconds, 65 ° C (1 ° C decrease per cycle), annealing at 20 seconds, and extension at 72 ° C, 30 seconds" for 10 cycles, "95 ° C. , 10 seconds of denaturation, 55 ° C., 20 seconds of annealing, and 72 ° C., 30 seconds of elongation "for 20 cycles (72 ° C., with 10 minutes of final elongation). The 10 μl reaction mixture for PCR includes 50 ng of genomic DNA as a template, 1 × GoTaq (R) Green Master Mix, and 0.5 μM specific primers HvNiR-GPspeF (5'-CCCGCATGCATATCCCATAG-3' / SEQ ID NO: 197). ) And HvNiR-GPspeR (5'-CCCGGACTAGTCCAAGATAC-3'/ SEQ ID NO: 198). PCR products were analyzed by electrophoresis in a TBE running buffer using a 1.5% agarose gel (Wako, Japan).

(6) Detection of distorted separation A chi-square (χ2) test was performed. Significant segregation distortion is shown using not only the estimated dominant ratio of 3: 1 or 1: 3 (df = 1), but also the Mendelian ratio of 1: 2: 1 (df = 2) expected for the null hypothesis. The genomic region was identified. Microsoft (R) Excel (R) was used for all calculations, charts, and color-coded diagrams.

[result]
(1) Creation of transformed HN × GP plant Prior to this, the present inventor resulted from a cross between GP and HN (with three backcrosses to HN as an explant for transformation). Attempts were made to use immature embryos of the BC3F8P recombinant chromosomal replacement line, but no transformed plants were obtained.

Here, F2 immature embryos generated from the cross between HN and GP (HN × GP) are agrobacterium carrying the binary vector pIG121-Hm having the genes of GUS (β-glucuronidase) and HPT (Hygromycin Phospho Transferase). -Inoculated with Tumefaciens strain AGL1 (Hiei, Y. et al., (1994) Plant J., 6, 271-282.). From 3,030 immature embryos, 293 hygromycin-resistant calluses were observed after selection on induction / selection medium. Green shoots were regenerated from 64 of these calluses on the regeneration medium. A total of 60 independent plants gave rise to healthy roots on regenerated medium, which were transplanted into pots and grown in a controlled environment. DNA was isolated from the leaves of 60 candidate plants and then used for genotyping and PCR analysis. In this PCR analysis, all 60 candidate plants were PCR positive for the GUS and HPT transgenes. These data showing transgene integration suggested that all 60 candidates carry genetic factors from the GP that allow transformation by the Agrobacterium method.

(2) SNP marker analysis of transformed HN × GP plants
Genotypes of 60 transformed HN × GP plants from 384-SNP Illumina GoldenGate (R) from Barley oligonucleotide pool analysis 1 (Close, TJ et al., (2009) BMC Genomics, 10, 1-13.) Determined using a subset of 124 markers on the platform.

The genetic location of all markers in this analysis can be obtained from the consensus gene map (Close, T.J. et al., (2009) BMC Genomics, 10, 1-13.). Genotyping data for transformed HN × GP plants are shown in Tables 1-9 above, and genotypes based on the order of markers on each chromosome are shown in Figure 1. Each transformed HN × GP plant had a unique genotype, a fact suggesting that these 60 plants arose from independent transformation events.

(3) Allele frequency and separation strain analysis Allele frequency was calculated based on genotyping data and mapped to the corresponding chromosome (Fig. 2A). Mendel's laws of independence predict that GP homozygous, heterozygous, and HN homozygous alleles will separate in a ratio of 1: 2: 1. GP alleles are located in the region between markers 2580-1645 (position: 55.0cM) and 3256-1196 (120.8cM) on chromosome 2H and between 4105-1417 (46.3cM) and 8020-87 (88.8cM) on chromosome 3H. In this region, the frequency exceeded 40% (Fig. 2A).

A chi-square (χ2) test was used to identify the segregation strains caused by selection for transformation (FIGS. 2B and 2C). Χ2 values for the predicted Mendelian ratio (HN: HE: GP = 1: 2: 1) were plotted at marker positions on the barley genome (Fig. 2B). The maximum χ2 value was 52.1, which was accompanied by the marker 4105-1417 on chromosome 3H. On the other hand, the peripheral region between 4105-1417 (46.3 cM) and 8020-87 (88.8 cM) showed statistically significant separation distortion (df = 2, p <0.01). The present inventor has named this region TFA1 (TransFormation Amenability 1). Other regions with significant segregation strain were observed between 2580-1456 (55.0 cM) and 3256-1196 (120.8 cM) on chromosome 2H (df = 2, p <0.01). This region contains two peaks with markers 6117-1507 (82.8cM, χ2 = 22.9) and 7576-818 (117.9cM, χ2 = 13.6), and these loci were named TFA2 and TFA3, respectively. .. The increased frequency of GP alleles in these regions suggested that factors involved in transformation efficiency in GP were present there. Interestingly, an increased frequency of HN alleles was observed between 8377-1022 (30.99cM) and 4648-775 (34.25cM) on chromosome 5H and was named TFA4. The χ2 values of loci with significant segregation distortion are summarized in Tables 1-9.

To test the dominant orientation of the identified loci, the dominant segregation ratio (3: 1) was used in the χ2 test (Fig. 2C). Under the assumption that the GP allele is functionally recessive ([HN + HE]: [GP] = 3: 1), in addition to TFA1, TFA2, and TFA3, regions and markers showing significant distortion are chromosome 2H. Between markers 1685-396 (21.6cM) and 7032-201 (29.2cM), 7818-967 (150.4cM) on chromosome 3H, and ABC14522-1-8-350 (5.5cM) and 2055 on chromosome 4H. Observed between -947 (21.6 cM). These added loci were named TFA5, TFA6, and TFA7, respectively (line in Figure 2C). On the other hand, under the assumption that the GP allele is dominant ([GP + HE]: [HN] = 3: 1), along with TFA1, TFA3, and TFA4, 4986-1214 (84.3cM) and 4564 on chromosome 4H. Between -604 (87.5cM), loci were identified between Consensus GBS0451-1 (155.1cM) on chromosome 5H and between Consensus GBS0346-1 (12.5cM) and 1769-545 (17.0cM) on chromosome 6H. These new loci have been named TFA8, TFA9, and TFA10, respectively (Figure 2C). Interestingly, the TFA2 χ2 value showed significant separation distortion under [HN + HE]: [GP] = 3: 1, but not under GP dominant conditions.

(4) Cloning and genotyping of HvNiR gene In order to test whether the barley ortholog of rice Nir is involved in the transformation efficiency, the present inventor made these for comparison between HN and GP. NiR orthologs were isolated from two cultivars. The putative HvNiR gene containing the expected start and stop codons was 2,965 bp for HN and 3,097 bp for GP. Analysis of the HvNiR gene in HN and GP revealed 4 exons and 3 introns, and comparison of DNA sequences between HN and GP found 14 SNPs in introns and 3 synonymous substituted SNPs in exons. (Fig. 3A). In addition, the HN allele held 7 bp and 23 bp insertions in the first intron, while the GP allele held 161 bp insertions homologous to the retrotransposon-like sequence in the first intron (Fig. 3A). Despite these differences, the expected amino acid sequences of HvNiR in HN and GP were identical.

To investigate whether insertion of HvNiR in the first intron is involved in transformation efficiency, a codominant sequence tag site (STS) marker that amplifies the region across GP-specific 161 bp insertions for genotyping It was designed and STS analysis was performed using DNA from 60 transformed HN × GP plants. Allele analysis reveals a segregation ratio of 12:37:11 (HN: HE: GP), which results in a single-factor Mendelian ratio without segregation distortion (Fig. 3B, 1: 2: 1 ratio χ2 = 3.3). Fits.

(5) Genome-wide locus-locus interactions Usually, populations with conflicting properties (eg, resistance vs. resistance to given stress) are used to analyze locus-locus interactions. We have developed transformed plants to map transformation efficiencies, but because all strains are transform sensitive, our population is selective biased and standard software. Is not useful for detecting locus-locus interactions. To analyze locus-locus interactions in transformed HN × GP plants, we group genotypes by allelic type of a single marker, followed by other markers associated with the first marker. The marker separation ratio and the χ2 value were calculated. For example, using the HvNiR locus, HN × GP plants were divided into a group of 11 plants for GP, a group of 37 plants for HE, and a group of 12 plants for HN based on the allele in HvNiR (above in Figure 4A, respectively). , Center, bottom panel), and then the allele ratio (band chart) at each of the other loci and the p-value of the χ2 test (heat map, df = 2) were compared. Since HvNiR is located on the long arm of chromosome 6H, the frequency of alleles in this region was consistent with the frequency of HvNiR. On chromosome 2H, GP allele-biased strain was observed around TFA2 in the GP- and HN-type HvNiR groups, but not in the HE-type group (Fig. 4A). Around TFA3, GP and HE alleles were also dominant in the HN- and HE-type HvNiR groups, but not in the GP type group. Interestingly, there was no segregation distortion around TFA1 among GP-type HvNiR plants. Further segregation distortion was also observed on the long arm of chromosome 4H in GP-type HvNiR plants (Fig. 4A).

Using the method described above, χ2 testing of all non-redundant markers (111 in total) was performed on transformed HN × GP plants. First, we calculate the number of allele combinations between one locus (locus A) and another (locus B) (eg GP-HN for locus AB). Then, the χ2 value (df = 2) for the strain of locus B was calculated between alleles of each type of locus A. FIG. 4B is a color-coded diagram of the P-value representing the significant separation distortion of locus B on the horizontal axis relative to locus A on the vertical axis, GP (left panel), HE (center panel), and HN ( It is divided for each allele in the right panel). Separation strain was observed around TFA1 as locus B for almost all markers. However, no segregation distortion was observed in the region of chromosome 6H containing the HvNiR in which the HN × GP plant had the GP allele (left panel in FIG. 4B). Plants with GP alleles in this region of chromosome 6H showed segregation strain not only in TFA2 but also in the long arms of chromosome 4H (left panel in Figure 4B). Apart from these loci, 1H-4H, 2H-7H, 3H-7H, 4H-5H, 7H-2H, and 7H- when the major segregation strain is accompanied by a GP allelic marker as locus A. Observed at 4H (right panel in Figure 4B). When accompanied by an HN allelic marker as locus A in HN × GP plants, segregation strains were 1H-2H, 1H-5H, 2H-6H, 3H-2H, 6H-2H and 7H-2H in addition to TFA1. Observed (right panel in Figure 4B). In particular, plants with HN alleles at chromosomes 6H and 7H as locus A showed extensive segregation distortion at chromosome 2H as locus B.

(6) Verification of genetic factors related to transformation susceptibility by alternative crosses In order to verify TFAs, in particular TFA1, TFA2, and TFA3 as important genetic factors for transformation in caterpillars, the present inventor used Morex ( F2 immature embryos derived from the cross between MO) and Golden Promise (MO × GP) were transformed by the Agrobacterium method.

Specifically, an Agrobacterium infection experiment was conducted on immature embryos (F2 generation) of first-generation (F1) hybrids of "Morex" and "Golden Promise". Immature embryos infected with Agrobacterium were co-cultured for 3 days, placed in a medium containing no antibiotics for 7 days, and cultured without selection. Then, resistant callus was selected using a selection medium containing hygromycin. The selection was conducted twice for 14 days (28 days in total). Only apparently proliferated callus were subcultured in redifferentiation medium. Subculture with fresh redifferentiation medium every 14 days and callus that did not redifferentiate after 42 days was discarded. The redifferentiated shoots were transferred to a hormone-free medium for rooting and potted. Two independently transformed MO × GP plants from 1,722 immature embryos were obtained and genotyped using the GoldenGate (R) platform. The results are shown in Tables 10-12.

The TFA1, TFA2, and TFA3 regions corresponded to homoGP and heterogenotypes in both plants. These facts confirm that the TFA1, TFA2, and TFA3 GP alleles are effective for transformation in alternative haplotypes of barley.

By selecting individual barley individuals using the genomic region involved in transformation sensitivity found in the present invention as an index, it is possible to increase the transformation efficiency of all barley varieties. Therefore, the present invention can contribute significantly to the breeding of barley, in particular.

SEQ ID NOs: 1-190
<223> SNP
SEQ ID NOs: 191-198
<223> Primer sequence

Claims (4)

The golden promise is crossed with any barley variety, and in the obtained progeny, the presence of at least one genomic region (a) to (c) below is detected, and the genomic region is homozygous or heterozygous and golden promise type. A method for producing barley that is sensitive to transformation by the Agrobacterium method, including selection of individuals.
(a) Genome region sandwiched between the marker NIASHv1109O03_00000798_3H and the marker NIASHv1150F05_00000946_3H on chromosome 3H
(b) Genome region sandwiched between the marker NIASHv2027F11_00000300_2H and the marker NIASHv2016D11_00000337_2H on chromosome 2H
(c) Genomic region sandwiched between the marker NIASHv2108A23_00000279_2H and the marker FLOUbaf102a14_00001505_2H on chromosome 2H The golden promise is crossed with any barley variety, and in the obtained progeny, the presence of at least one marker (a) to (c) below is detected, and an individual whose marker is homo or hetero and golden promise type is detected. A method for producing barley that is sensitive to transformation by the Agrobacterium method, including selection.
(a) A marker selected from the group consisting of the marker NIASHv1109O03_00000798_3H on chromosome 3H, the marker NIASHv1150F05_00000946_3H, and the markers existing in the genomic region sandwiched between these markers.
(b) A marker selected from the group consisting of the marker NIASHv2027F11_00000300_2H on chromosome 2H, the marker NIASHv2016D11_00000337_2H, and the markers existing in the genomic region sandwiched between these markers.
(c) A marker selected from the group consisting of the marker NIASHv2108A23_00000279_2H on chromosome 2H, the marker FLOUbaf102a14_00001505_2H, and the markers existing in the genomic region sandwiched between these markers. Comprising the following (a) ~ (c) at least one pair of oligonucleotides designed so as to sandwich the marker, a reagent for testing a susceptibility to transformation by Agrobacterium method in barley, the marker A reagent for examining whether or not a homozygous or heterozygous is a golden promise type, and if it is a golden promise type, determining that it is sensitive to the transformation .
(a) a marker in the chromosome 3H NIASHv1109O03_00000798_3H, marker selected from the group consisting of marker present in the genome region between the markers NIASHv1150F05_00000946_3H, and their markers
(b) A marker selected from the group consisting of the marker NIASHv2027F11_00000300_2H on chromosome 2H, the marker NIASHv2016D11_00000337_2H, and the markers existing in the genomic region sandwiched between these markers.
(c) A marker selected from the group consisting of the marker NIASHv2108A23_00000279_2H on chromosome 2H, the marker FLOUbaf102a14_00001505_2H, and the markers existing in the genomic region sandwiched between these markers. A reagent for testing susceptibility to transformation by the Agrobacterium method in Omugi, which comprises an oligonucleotide that hybridizes to a nucleotide sequence on the genome containing at least one of the following markers (a) to (c) . A reagent for examining whether or not the marker is homozygous or heterozygous and has a golden promise type, and if it is a golden promise type, determining that the marker is susceptible to the transformation .
(a) A marker selected from the group consisting of the marker NIASHv1109O03_00000798_3H on chromosome 3H, the marker NIASHv1150F05_00000946_3H, and the markers existing in the genomic region sandwiched between these markers.
(b) A marker selected from the group consisting of the marker NIASHv2027F11_00000300_2H on chromosome 2H, the marker NIASHv2016D11_00000337_2H, and the markers existing in the genomic region sandwiched between these markers.
(c) A marker selected from the group consisting of the marker NIASHv2108A23_00000279_2H on chromosome 2H, the marker FLOUbaf102a14_00001505_2H, and the markers existing in the genomic region sandwiched between these markers.

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