RU2787519C2 - Method for detection of atypical brassica oleracea plant - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology, in particular it proposes a method for detection of an aneuploid Brassica oleracea plant, including: PCR in real time, using, as a matrix, DNA isolated from a sample obtained from Brassica oleracea plant for testing and DNA markers specific for each of two or more chromosomes of Brassica oleracea plant; and isolation of chromosomic aneuploidy by a relative difference in amplification amount between obtained DNA markers.

EFFECT: according to one of options of implementation of the present invention, it is possible to simply, accurately, and quickly detect an atypical plant (chromosomic aneuploid), which can be found in Brassica oleracea varieties, in a laboratory equipped with general molecular-biological equipment during quality control of seeds and breeding researches.

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Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is based on the prior Japanese Patent Application No. 2017-157384, filed August 17, 2017, the entire content of which is incorporated herein by reference, and asserts its priority.

BACKGROUND OF THE INVENTION

Technical field

The present invention relates to a method for detecting an atypical plant (chromosomal aneuploidy) Brassica oleracea. More specifically, the present invention relates to a method that is capable of accurately and rapidly classifying and detecting individuals exhibiting various abnormal morphology associated with aneuploidy in Brassica oleracea (hereinafter, hereinafter, may be abbreviated as "B. oleracea" ) by molecular biology. ) at any stage of growth.

Prior Art

Brassicaceae plants are plant species that originated in the Middle East and the Mediterranean coast. This family includes plants of the genus Brassica, which are extremely important agricultural crops. The plant species Brassica oleracea is very important and includes, as non-limiting examples, B. oleracea var. capitata (cabbage), B. oleracea var. italica (broccoli), B. oleracea var. botrytis (cauliflower), B. oleracea var. gemmifera (Brussels sprouts), B. oleracea var. gongyloides (kohlrabi), B. oleracea var. acephara (ornamental cabbage, browncol), and B. oleracea var. albograbra (Chinese cabbage).

In cultures of Brassica oleracea, commercial breeding is most often directed to hybrid plants (F1) of the first generation, using the properties of self-incompatibility (SI) or cytoplasmic male sterility (CMS). Compared to native varieties or OP (open pollinated), F1 varieties show excellent environmental adaptability and high uniformity. Thus, these varieties have a high commercial value and are used in many countries.

It has been published that individuals showing a morphology other than normal morphology appear with a certain frequency even if they are F1 varieties inheriting parental genes (see V. Ruffio-Chable et al., ISHS Acta Horticulturae 539 (2000) p 89. Developmentally "Aberrant" plants in F1 hybrids of Brassica oleracea (Non-Patent Document 1)). As a rule, such atypical individuals have an extremely low value, like agricultural products, and usually cannot be sent as fresh products. If a large number of individuals exhibiting an atypical phenotype include certain seeds or varieties, this can lead to a major commercial problem. Thus, seed companies may have to abandon such seeds.

The reason for the appearance of such atypical species was not known for a long time, but scientists have suggested that there were environmental influences during seed production or when growing a crop, or there were influences such as mutation or epigenetic changes.

The above problem of off-type plants also greatly hinders the quality control function of agricultural seed production enterprises. Seed companies that produce and market F1 hybrid seeds conduct testing using polymorphic DNA markers or isozymes to verify seed varietal purity before commercializing these products. However, these atypical individuals have the same F1 genotype as the variety and cannot be detected by such laboratory tests. For this reason, the development of a rapid testing method for non-typical plants has been urgently needed for a long time.

In addition, in breeding studies, it is necessary to improve varietal characteristics so that such atypical individuals are not often encountered. However, it is difficult to identify atypical plants by their appearance at the seedling stage. For this reason, in order to accurately calculate the frequency of off-types, it was necessary to grow the plants on a large scale in the field and have the characteristics of the individuals carefully assessed by an experienced breeder. However, such a test tends to be subjective and there is concern that the result may vary depending on the person who performs the assessment. In addition, it is difficult for even a skilled breeder to make an accurate judgment, and results may vary depending on the growth stage and growing environment. In light of this, seed companies have long wanted a simple test method for seedlings.

For example, V. Chable et al., Euphytica 170 (2009) p275, "Aberrant" plants in cauliflower: 2. Aneuploidy and global DNA methylation (Non-Patent Document 2) posits the possibility that the abnormal morphology appearing in the F1 cauliflower cultivar , comes from aneuploidy. Several experimental studies have reported the use of the flow cytometer as a general way to detect aneuploidy.

However, it is not easy to accurately determine a single chromosome difference in the flow cytometer method, and even if an endogenous control is added to the sample, it can be difficult to make a decision, as shown in the data described in N. Roux et al., Plant cell Report 21 (2003) p483, Rapid detection of aneuploidy in Musa using fluid cytometry (Non-Patent Document 3). There is also a difference in chromosome size, and the ratio of the genomes of the largest chromosome to the smallest chromosome can be as high as almost 2:1. In the case where a relatively small chromosome is added, it is difficult to judge because the difference between the peaks of normal individuals and aneuploids is extremely small, and the detection sensitivity has a great influence on the accuracy of the results.

For this reason, the method that uses the flow cytometer creates problems such as the inability to perform a large scale test in a conventional laboratory.

In addition, in the flow cytometer method, even if a highly reliable experimental system is assembled, in the case of plants that have aneuploidy in multiple chromosomes. For example, in the case of an aneuploid in which a trisomy of chromosome 1 and a monosomy of chromosome 2 are combined, and thus 18 chromosomes are contained in the nucleus, it is defined as a normal individual.

In addition, it is difficult to determine which chromosome caused aneuploidy in a simple test using a flow cytometer. Depending on the chromosome with trisomy, there are also species in which fresh produce can be harvested without quality problems; although the maturity of the plant varies somewhat. For example, in the case of trisomy of chromosomes 2 and 7 in broccoli and trisomy of chromosomes 1, 2 and 7 in cauliflower, thus in some cases the presence of an aneuploid is not immediately associated with a decrease in market value. For this reason, it is important to be able to distinguish the type of trisomy individually in the field. From a breeding point of view, the fact that any chromosome tends to trisomy provides important information. In this regard, there was a need to develop a simple testing method that can analyze detailed aneuploidy for each chromosome.

So far, methods have been reported for distinguishing genotypes in plants by PCR using DNA markers. For example, JP 3836451 B2 (Patent Document 1) discloses a method for determining the genotype involved in the production of the pungent ingredients of Capsicum plants. However, to the best of the present inventors' knowledge, no method has been reported to date for testing atypical Brassicaceae plants.

[List by prior art]

patent document

Patent Document 1: Japanese Patent Publication No. 3836451 (JP 3836451 B2)

non-patent document

Non-Patent Document 1: V. Ruffio-Chable et al., ISHS Acta Horticulturae 539 (2000) p89, Developmentally "Aberrant" plants in F1 hybrids of Brassica oleracea.

Non-Patent Document 2: V. Chable et al., Euphytica 170 (2009) p275, "Aberrant" plants in cauliflower: 2. Aneuploidy and global DNA methylation.

Non-Patent Document 3: N. Roux et al., Plant cell Report 21 (2003) p483, Rapid detection of aneuploidy in Musa using fluid cytometry.

Non-Patent Document 4: I. Parkin et al., Genetics 171 (2005) p765, Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana.

Non-Patent Document 5: X. Cheng et al., Theoretical Applied Genetics 118 (2009) p1121, Development and genetic mapping of microsatellite markers from genom survey sequences in Brassica napus.

[SUMMARY]

Problems to be solved by invention

The aim of the present invention is to provide a method for accurately and quickly detecting an atypical plant (chromosomal aneuploid) that may appear in the species Brassica oleracea in order to solve the problem described above affecting the course of seed quality control and breeding research. In addition, the method can be carried out in a laboratory equipped for general molecular biological methods.

Ways to solve the problem

As a result of extensive research carried out to meet the requirements of scientists for seed quality control and breeding, the authors of the present invention were able to accurately and quickly identify aneuploids of all chromosomes in Brassica oleracea species using real-time PCR. This method is easy to perform in a laboratory equipped for real-time PCR, and it has made it possible to easily detect atypical plants. In addition, this method is able to accurately and quickly classify and detect individuals exhibiting various abnormal morphologies caused by aneuploidy at any stage of growth using a molecular biological method.

Thus, according to the present invention, the following inventions are offered:

<1> A method for detecting an aneuploid of a Brassica oleracea plant, comprising:

performing real-time PCR using, as a template, DNA isolated from a sample obtained from a Brassica oleracea plant for testing, and DNA markers specific for each of two or more chromosomes of a Brassica oleracea plant; and detection of chromosomal aneuploidy by the relative difference between the amplification values obtained using DNA markers.

<2> The method according to <1>, comprising determining whether a test plant is an aneuploid in a chromosome from all chromosomes, using DNA markers specific for each of chromosomes 1 to 9 of a Brassica oleracea plant.

<3> The method according to <1> or <2>, wherein the method uses as a DNA marker a primer that is specific to only one of the chromosomal DNA of Brassica oleracea plant and can produce an amplification product in a PCR reaction when a chromosomal DNA.

<4> The method according to any one of <1> to <3>, wherein the method uses as DNA markers (i) a primer that is specific for only one of the chromosomal DNA of the Brassica oleracea plant and can produce an amplification product in PCR reactions when chromosomal DNA is present; and (ii) a probe that is specific for chromosomal DNA identical to any of the chromosomal DNAs described in (i) and can detect an amplification product obtained by a PCR reaction based on the primer described in (i).

<5> The method according to <3> or <4>, wherein the method uses an intercalator that binds to a double-stranded DNA synthesized by a PCR reaction and emits fluorescence, or uses a probe modified with a fluorescent dye to emit fluorescence upon reaction elongation PCR.

<6> The method according to any one of <1> to <5>, wherein the method uses an increase in a fluorescent signal obtained by real-time PCR as an indicator for detecting an aneuploid chromosome.

<7> The method according to any one of <1> to <6>, wherein the method uses one or more primers with the nucleotide sequences shown in SEQ ID NOs: 1 to 18 as a DNA marker.

<8> The method according to any one of <1> to <7>, wherein the method uses, as a DNA marker, one or more primers with the nucleotide sequences shown in SEQ ID NOs: 1 to 18 and one or more probes with the nucleotide sequences shown in SEQ ID NO: 19 to 27.

<9> A primer set for detecting an aneuploid Brassica oleracea plant, comprising at least one or more primers with the nucleotide sequences shown in SEQ ID NOs: 1 to 18.

<10> A set of primers and probes for detecting an aneuploid plant Brassica oleracea, comprising at least one or more primers with the nucleotide sequences shown in SEQ ID NO: 1 to 18; and at least one or more fluorescent dye-modified probes with the nucleotide sequences shown in SEQ ID NOs: 19 to 27.

<11> A Brassica oleracea culture breeding method comprising estimating a chromosome aneuploid frequency for each genetic line of a Brassica oleracea plant using the method of any of <1> to <8> to select a line with a low aneuploid rate.

<12> A method for quality control of Brassica oleracea seeds, comprising testing to determine the degree of contamination with aneuploids contained in the seeds of a batch of Brassica oleracea seeds, using the method of any one of <1>-<8>

<13> A quality control method for Brassica oleracea plants, comprising testing to determine the degree of contamination by aneuploids contained in Brassica oleracea plants, using the method of any one of <1>-<8>.

Invention Effects

According to the test method of the present invention, it becomes possible to easily and accurately assess the frequency of occurrence of off-types and the future morphology of each of the detected aneuploids, which makes it possible to check the purity of the variety of commercial seed lots and the frequency of occurrence of off-types in breeding lines. As a result, it becomes possible to efficiently, quickly and in the shortest possible time to provide ways for stable supply of high quality commercial seeds and breeding lines with good characteristics.

[SHORT DESCRIPTION OF PICTURES]

FIG. 1 shows aneuploid phenotypes from broccoli plants, where (A) represents a normal individual (Normal), (B) represents trisomy on chromosome 1 (+C1), (C) represents trisomy on chromosome 2 (+C2), (D) trisomy 3 (+C3) present, (E) trisomy 4 (+C4) present, (F) trisomy 5 (+C5) present, (G) trisomy 6 (+C6), ( H) represents trisomy 7 (+C7), (I) represents trisomy 8 (+C8), and (J) represents trisomy 9 (+C9).

FIG. 2 shows an image of an amplification curve when performing quantitative analysis by real-time PCR, where the figure is based on the example of trisomy 6, and where the amount of DNA as a template added to the PCR reaction solution is unchanged. For example, in the case of trisomy on chromosome 6 (Trisomy), the amplification curve of a marker located on chromosome 6 rises faster than in a normal individual (Normal).

FIG. 3 shows an example of aneuploid detection, namely an example of a result calculated from the relative difference between amplification values obtained with DNA markers when performing real-time PCR. Ninety-six individuals derived from the F1 generation of the broccoli cultivar used as materials were tested by multiplex PCR using a marker on chromosome 4 and a marker on chromosome 6. The marker on chromosome 6 shows the relative differences between amplification values with chromosome 4 as standard, and as a consequence, trisomy 6 and trisomy 4 can be identified.

FIG. 4 shows photomicrographs of chromosomes observed in pollen mother cells of trisomic plants. Using microscopic observation of cells in the primary metaphase of meiosis of pollen mother cells, in the case of a normal chromosome, nine bivalent chromosomes (2n=18) were observed. While at the chromosomal level in a trisomic plant, nine bivalent chromosomes and one additional chromosome (indicated by an arrow) (2n+1) were observed.

FIG. 5 shows aneuploid phenotypes from cauliflower plants, where (A) represents a normal individual (Normal), (B) represents trisomy 1 (+C1), (C) represents trisomy 2 (+C2), (D ) shows trisomy 4 (+C4), (E) shows trisomy 6 (+C6), (F) shows trisomy 7 (+C7), and (G) shows trisomy 9 (+C9 ).

FIG. 6 shows aneuploid phenotypes from cabbage plants, where (A) represents a normal individual (Normal), (B) represents trisomy on chromosome 1 (+C1), (C) represents trisomy on chromosome 2 (+C2), (D) trisomy 4 (+C4) present, (E) trisomy 5 (+C5) present, (F) trisomy 6 (+C6) present, (G) trisomy 7 (+C7) present, ( H) represents trisomy on chromosome 8 (+C8), (I) represents trisomy on chromosome 9 (+C9), and (J) represents an individual with aneuploidy on chromosome 1 and chromosome 2 (+C1, +C2), on ( K) represents an individual with aneuploidy on chromosome 1 and chromosome 8 (+C1, +C8), and (L) represents an individual with aneuploidy on chromosome 5 and chromosome 8 (+C5, +C8).

FIG. 7 shows the phenotypic characteristics of aneuploids derived from broccoli, cauliflower and cabbage plants. These characteristics refer only to some of the phenotypic characteristics observed in the study for this example, and it is not necessarily possible to generalize and characterize the distinguishing features for the respective plant species and aneuploids.

[EMBODIMENTS]

Hereinafter, the present invention will be described in detail.

As described above, the present invention relates to a method for detecting an aneuploid of a Brassica oleracea plant, including performing real-time PCR using, as a template, DNA isolated from a sample obtained from a test plant Brassica oleracea and DNA markers specific for each of two or more chromosomes of the plant Brassica oleracea; and detection of chromosomal aneuploidy by the relative difference between the amplification values obtained using DNA markers. According to a preferred embodiment of the present invention, the number of chromosomes used for the detection method is 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, and 8 or more are more preferred in that order. It is most preferred to use markers for all nine chromosomes.

Thus, the method of the present invention preferably comprises: determining whether a test plant is chromosomal aneuploid using DNA markers specific for each of chromosomes 1 to 9 of the Brassica oleracea plant.

In the present invention, a Brassica oleracea plant means a plant of the Brassica oleracea species of the genus Brassica, which, as non-limiting examples, includes B. oleracea var. capitata (cabbage), B. oleracea var. italica (broccoli), B. oleracea var. botrytis (cauliflower), B. oleracea var. gemmifera (Brussels sprouts), B. oleracea var. gongyloides (kohlrabi), B. oleracea var. acephara (ornamental cabbage, browncol), B. oleracea var. albograbra (Chinese cabbage). In the present invention, the Brassica oleracea plant is broccoli, cauliflower or cabbage.

The term "aneuploid" or "atypical" as used herein means an individual with an abnormal number of chromosomes (aneuploidy). In this case, there is an anomaly in the number of chromosomes found in the Brassica oleracea plant. The number of chromosomes in a normal Brassica oleracea plant is eighteen (2n=18), thus resulting in nine pairs of chromosomes in one cell. For example, in the case of an aneuploid, in particular an aneuploid plant with trisomy for chromosome 1, the number of chromosomes 1 increases from two to three; this indicates that the ploidy level of chromosome 1 is impaired. Although some aneuploid individuals may be acceptable as agricultural products or fresh products depending on the plant species and growing conditions, most often atypical individuals are of low value as agricultural products and in many cases may not be sold as fresh products. For this reason, it is important to be able to quickly and easily identify aneuploids.

In the aneuploid detection method of the present invention, a sample of a Brassica oleracea test plant is taken, DNA is isolated from a sample obtained from a Brassica oleracea test plant, and the DNA is used as a PCR template.

With regard to the DNA extraction method, any method known to the person skilled in the art can be used to isolate the nucleic acid. In the present invention, the isolated crude DNA can be used as a template for PCR. For example, a phenol/chloroform method, a detergent cell lysis method, a protease enzyme cell lysis method, a glass bead physical disruption method, a repeated freeze/thaw treatment method, and a combination thereof can be used for DNA extraction. It is also possible to use various DNA extraction kits sold by reagent manufacturers, such as the QIAGEN Plant mini kit (manufactured by QIAGEN GmbH). DNA isolated by these methods is preferably in a state suitable for use as a template for PCR. For example, DNA is preferably stored at low temperature after being dissolved in an appropriate buffer solution. The purity of the obtained DNA can be checked by measuring the optical density at 230, 260 and 280 nm using a spectrophotometer. When performing PCR, it is preferable that the absorbance ratio at 260/230 nm is 2.0 or more, and the absorbance ratio at 260/280 nm is from 1.8 to 2.0. With regard to the resulting DNA solution, it can be confirmed that the amplification occurs by PCR using a primer pair for an endogenous gene common to plants.

In the detection method of the present invention, real-time PCR is carried out using, as a template, DNA isolated from a sample obtained from a Brassica oleracea plant to be tested and a DNA marker capable of amplifying a portion of each chromosome of the Brassica oleracea plant. In particular, real-time PCR is carried out using as a DNA marker two or more DNA markers specific for each of the two or more chromosomes of the Brassica oleracea plant. More specifically, real-time PCR is carried out using as a DNA marker two or more DNA markers specific for each of two or more chromosomes from chromosomes 1 to 9 of the Brassica oleracea plant.

The DNA marker used herein is not particularly limited as long as it is a DNA marker located on any of the chromosomes of the Brassica oleracea plant that is present as a single copy in the genome and is not affected by other sequences present on different chromosomes. The present invention utilizes such a marker, thereby allowing detection of the relative difference between amplification values obtained with DNA markers by real-time PCR, and testing of aneuploids from different cultures of Brassica oleracea.

Thus, it is preferred that the marker is a primer pair that is specific for only one of the chromosomal DNA of the Brassica oleracea plant and can produce an amplification product by a PCR reaction when chromosomal DNA is present. Here, the term "specific for only one chromosomal DNA of the plant Brassica oleracea" means that the primer is specific for only one of the nine pairs of chromosomes, but not specific for other chromosomes. When there is chromosomal DNA specific for only one of the nine pairs of chromosomes, the primer can amplify the target product through a PCR reaction. For example, the DNA marker used may include a primer that is specific for chromosome 1 DNA only and can produce an amplification product by a PCR reaction when chromosome 1 DNA is present.

With regard to the design of the primer to be used, one skilled in the art can appropriately prepare a primer for use with only one of the chromosomal DNA of the Brassica oleracea plant so that it can produce an amplification product by the PCR reaction when chromosomal DNA is present. In particular, one skilled in the art can appropriately prepare the desired primer by referring to the description of Example 1 below.

In the present invention, it is possible to test an aneuploid by performing real-time PCR using a primer having the above properties, and the intercalator method can be used as one of the preferred embodiments. In the intercalator method, the intercalator that has been added to the PCR reaction solution binds to the double-stranded DNA synthesized by the PCR reaction, and the intercalator emits fluorescence. Thus, an elongation reaction with the primer occurs to amplify the product, thereby resulting in fluorescence. This fluorescence is detected, which makes it possible to measure the relative difference between amplification values obtained using DNA markers.

For example, SYBR Green I can be used as an intercalator.

According to a specific embodiment of the intercalator method, (i) a primer pair that is specific for only one of the chromosomal DNA of the Brassica oleracea plant and can amplify the product through a PCR reaction when chromosomal DNA is present, and (ii) the intercalator is added to the reaction solution PCR; The fluorescence signal emitted in the elongation reaction step of the PCR is measured for each cycle, and the amplification values obtained from the DNA markers are calculated. Thus, it is possible to measure the relative difference between amplification values obtained with DNA markers.

Another preferred embodiment of the present invention is a probe method using a probe capable of detecting an amplification product generated by a PCR reaction based on a primer having the above properties. The probe to be used is not particularly limited as long as the probe can detect the target amplification product, and it is preferable to use a labeled probe that is modified with a fluorescent dye so that fluorescence is emitted by the elongation reaction in PCR. A method for calculating a relative difference between amplification values obtained with DNA markers is carried out by measuring the fluorescence emitted during an elongation reaction in PCR, whereby the method can be carried out in the same principle as the intercalator method. In addition, the probe method compared to the intercalator method may reduce the risk of detecting non-specific PCR products.

According to a specific embodiment of the probe method, (i) a primer pair that is specific for only one of the chromosomal DNA of the Brassica oleracea plant and can amplify the product via a PCR reaction when chromosomal DNA is present; and (ii) a probe that is specific for chromosomal DNA identical to any of the chromosomal DNA described in (i) and can detect the amplification product in a PCR reaction based on the primers described in (i); the fluorescence signal emitted in the elongation reaction step of the PCR is measured for each cycle, and the amplification values obtained by the DNA markers are calculated. Thus, it is possible to measure the relative difference between amplification values obtained with DNA markers.

As for the fluorescently labeled probe, a TaqMan probe doubly labeled with a fluorescent substance and a quencher is preferred. In the TaqMan probe, the 5' end of the nucleic acid of the probe is typically modified with a fluorescent agent (fluorescent reporter dye) and the 3' end is typically modified with a quenching agent (fluorescent quenching dye). Examples of the reporter fluorescent dye include: fluorescein-based fluorescent dyes such as Cy3, Cy5, 6-carboxyfluorescein (6-FAM), 6-carboxy-4,7,2',7'-tetrachlorofluorescein (TET), and 6-carboxy -2',4',7',4,7-hexachlorofluorescein (HEX). As for the quenching fluorescent dye, a rhodamine-based fluorescent dye such as 6-carboxytetramethylrhodamine (TAMRA) or 6-carboxy-X-rhodmine (ROX) can be used. In the case of multiplex PCR, a dark quencher such as BHQ-1, BHQ-2, BHQ-3 or Eclipse is preferred. In the present invention, for example, a hydrolysis probe labeled with three types of fluorescent dyes such as FAM, HEX and Cy5 at the 5' end and two types of quenchers such as BHQ-1 and BHQ-3 at the 3' end can be used.

Thus, in the detection method of the present invention, it is preferable to use an intercalator that binds to double-stranded DNA synthesized by PCR reaction and emit fluorescence, or use a probe modified with a fluorescent dye to emit fluorescence during the elongation reaction in PCR.

In the case of using a probe modified with a fluorescent dye, multiplex PCR can be performed using probes labeled with different types of fluorescent dyes, mixing them with several types of markers. Similar to the method described above for each of the individual samples tested and each of the markers, the relative difference between amplification values obtained with DNA markers can be measured based on the fluorescent signals emitted by the fluorescent probes. In this method, in the case of multiplex PCR, it is possible to calculate the relative difference between the amplification values obtained with DNA markers in the same reaction solution as the endogenous control, and the reduction of experimental errors will increase the reliability of the result.

Thus, in accordance with another preferred embodiment of the present invention, multiplex PCR is carried out using the probe method.

Thus, according to one of the preferred embodiments of the present invention, the increase in the fluorescent signal obtained by real-time PCR is used as an indicator for the detection of a chromosome aneuploid in the detection method of the present invention.

An oligonucleotide to be used as a primer or probe can be synthesized by a method known in the art as an oligonucleotide synthesis method, such as the phosphotriethyl method or the phosphodiester method, using a conventional automated DNA synthesizer.

According to a more preferred embodiment of the present invention, the DNA marker used may be one or more primers with the nucleotide sequences shown in SEQ ID NO: 1 to 18, and more preferably, one or more of the primers described in Table 1 below can be used. According to another more preferred embodiment of the present invention, one or more primers with the nucleotide sequences shown in SEQ ID NO: 1 to 18 and one or more probes with the nucleotide sequences shown in SEQ ID NO: 19 to 27 can be used. More preferably , one or more of the primers described in Table 1 and one or more of the probes described in Table 2 can be used. These markers are markers corresponding to chromosomes 1 to 9.

As used herein, a DNA marker "having" a nucleotide sequence means that the marker has a nucleotide sequence. In the present invention, the DNA marker is specific for the DNA of a predetermined chromosome, as described above. Thus, provided that the DNA marker has the properties of the marker, one or more (e.g., one, two or three, one or two, and more than one) of the bases in the nucleotide sequence corresponding to the DNA may be substituted, deleted, added, or removed, or the DNA marker may be a sequence that contains a nucleotide sequence that partially matches the DNA and retains predetermined properties. In such a case, the term "having" can be rephrased as "including". In addition, in the case where substitution, deletion, addition or deletion of one base is allowed, the term "having" can be rephrased as "essentially consisting of".

According to another more preferred embodiment of the present invention, the DNA marker used may be one or more primer pairs with the nucleotide sequences shown in SEQ ID NO: 1 to 18, and more preferably, may be one or more primer pairs described in Table 1 below. According to another more preferred embodiment of the present invention, one or more primers with the nucleotide sequences shown in SEQ ID NO: 1 to 18 and a probe with nucleotide sequences shown in SEQ ID NO: 19 to 27 corresponding to the primer pairs can be used. More preferably, one or more of the primer pairs described in Table 1 and one or more of the probes described in Table 2 can be used.

According to an even more preferred embodiment of the present invention, a more accurate and reproducible test can be performed using DNA markers divided into the following three sets:

a chromosome 6 marker, a chromosome 4 marker, and a chromosome 2 marker;

chromosome 9 marker, chromosome 3 marker, chromosome 8 marker; and

chromosome 1 marker, chromosome 5 marker, and chromosome 7 marker.

In the present invention, in addition to using the above primer and probe pairs, real-time PCR can be performed. For example, the method may be based on conventional methods described in Real-Time PCR Experiment Guide in Experimental Medicine, Supplement Edition, (YODOSHA CO., LTD), Saiki RK, et al., Science, 230: 1350-1354 (1985) , and Plant PCR Experimental Protocol in Plant Cell Engineering, Supplement Edition, supervised by Ko Shimamoto and Takuji Sasaki (1995), and can be performed using a commercially available real-time PCR kit or real-time PCR instrument according to the attached instructions.

As a real-time PCR instrument, for example, LightCycler 480 System II (manufactured by Roche) or the like can be used. At present, for example, "Premix Ex Taq (Perfect Real Time)" (manufactured by Takara Bio Inc.) or the like can be used as a reaction reagent, but the use is not specifically limited to the materials mentioned above.

First, PCR is carried out using as a template a DNA sample obtained by the above method and using a predetermined primer or primer pair of the present invention. Amplification by PCR is not particularly limited, except that the above primers or primer pair are used, and amplification by PCR can be carried out according to a conventional method. Regarding the PCR conditions (such as temperature and time for each of the denaturation, annealing and elongation steps and the number of cycles) and the composition of the PCR reaction solution (such as the amount of template DNA, type of buffer solution, primer concentration, type and concentration of DNA polymerase, dNTP concentration and magnesium chloride concentration), one skilled in the art can appropriately based on preliminary experiments or the like. select and establish conditions under which PCR amplification products can be obtained with the desired high sensitivity by PCR using the primer or primer pair described above. In addition, a real-time PCR master mix in which DNA polymerase, dNTP concentration, and magnesium chloride concentration are approximately optimized is commercially available, so it can be used when needed.

As described above, the number of chromosomes in Brassica oleacea is eighteen (2n=18), so in the case of a normal plant, there are nine pairs of chromosomes in each cell. For example, in the case of an aneuploid plant with trisomy for chromosome 1, the number of chromosome 1 increases from two to three copies. When real-time PCR is performed using the DNA isolated from these plants as a template and the DNA markers described in Table 1 or Tables 1 and 2 in the case of accurate standardization of the amount of DNA added to the reaction solution for trisomy 1 , the amplification curve of a marker located on chromosome 1 increases in early cycles compared to a normal individual. That is, the lower cycle threshold is set (also abbreviated as "Ct value", that is, the intersection point of the marker amplification curve obtained from the fluorescence intensity and the cutoff line set using a certain standard).

As a specific example, as shown in FIG. 2, when the amount of DNA added to the PCR reaction solution as a template is constant, for example, in the case of trisomy on chromosome 6 (Trisomy), the amplification curve of the marker located on chromosome 6 grows faster than that of a normal individual (Normal).

However, it is difficult to accurately standardize the amount of DNA in a large number of samples at a real test site. Thus, instead of standardizing the amount of DNA, the amplification of chromosome markers is subject to relative quantification, whereby the number of each of the chromosomes can be estimated from the relative difference between the amplification values obtained for each of the DNA markers, and aneuploidy such as trisomy can be determined.

In order to widely apply this method to Brassica oleracea species, it is necessary to develop PCR primers in the general area for Brassica oleracea species lacking single nucleotide polymorphisms (SNPs). As a result of intensive research carried out by the inventors of the present invention, success has been achieved in the development of DNA markers that can be widely applied to Brassica oleracea species and which can be used to perform an effective test using multiplex PCR. This led to the invention of the detection method of the present invention.

Thus, in the above-described detection method of the present invention, real-time PCR is performed using a chromosome-specific DNA marker, and chromosomal aneuploidy is determined by the relative difference between the amplification values obtained by the DNA markers.

Here, the relative difference between amplification values obtained with DNA markers can be confirmed by performing real-time PCR on DNA markers specific for each of two or more chromosomes of the tested Brassica oleracea plant and comparing the amplification values for each of the chromosomal markers. Thus, when the relative amplification of multiple chromosomal markers is assessed and an abnormality is present on any of the chromosomes, there is a clear difference between marker amplification on an aneuploid chromosome and marker amplification on chromosomes with a normal level of ploidy. As a result, it becomes possible to detect the presence of aneuploids.

Amplification values obtained with DNA markers can be confirmed with a marker amplification curve. The marker amplification curve can be easily plotted from the fluorescence intensity measured by the PCR reaction.

In the case of measuring the relative difference between amplification values obtained using DNA markers, i.e. when performing relative quantitation, it is preferable to use the amplification curve obtained for each marker. For example, an intersection point between an amplification curve and a threshold line set with a certain standard is defined as a cycle threshold (Ct value), and each Ct value is compared with another Ct value of another chromosome, thereby estimating the number of target chromosomes. In terms of looking for more reproducible estimates of the number of chromosomes, the Ct value can be replaced by another value calculated by the second derivative method, which differentiates the amplification curve twice, and takes the cycle value at the position showing the maximum score.

In the present invention, with regard to the Ct value indicated for each of the individuals in the test sample and each of the markers, the value calculated by the second derivative method as described above is selected, and the copy number ratio between the target regions of the genome can be estimated by the ΔΔCt method based on the relative differences between amplification values obtained using DNA markers.

In other words, a part of the preferred embodiment of the detection method of the present invention can be specifically expressed as the following methods (a) or (b):

(a) a method comprising determining a Ct value for each of the individuals in the test sample and each of the markers from a signal emitted by a fluorescent dye such as SYBR Green I; and estimating the copy number ratio of the target genomic region based on the relative difference between the amplification values obtained with DNA markers by different methods, for example, the ΔΔCt method, where a fluorescent dye such as SYBR Green I is mixed with the PCR reaction solution, and chromosomal markers separately amplified by PCR; or

(b) a method comprising determining the Ct value of each of the individuals in the test sample and each of the markers by the fluorescence of the signal emitted by the fluorescent probe, and estimating the copy number ratio of the target genomic region based on the relative difference between the amplification values obtained using DNA markers by various methods , for example, by the ΔΔCt method, where multiplex PCR is carried out using probes labeled with different types of fluorescent dyes and mixing several types of markers.

Although method (a) can be performed with a relatively inexpensive reagent, separate PCR tests are performed for each chromosome. Thus, multiple PCR tests are needed for each plant. In method (b), it is necessary to synthesize a fluorescent probe, while the relative difference between amplification values obtained by DNA markers can be made with an endogenous control as a standard. Thus, the number of tests can also be reduced.

The primers, primer pairs and probes described above can also be obtained as a kit. The kit of the present invention may be any as long as the kit includes a primer or primer pair as described above, or at least a primer or primer pair and a probe. Optionally, the kit may include a DNA molecule containing a target sequence as a positive control for PCR, a DNA extraction reagent, a PCR buffer, a PCR reagent such as DNA polymerase, a labeling agent for the label, and a guide.

Thus, according to another preferred embodiment of the present invention, a set of primers for detecting an aneuploid of a Brassica oleracea plant is provided, comprising at least one or more types of primers with the nucleotide sequences shown in SEQ ID NOs: 1-18. Additionally, in yet another preferred embodiment, the present invention provides a set of primers and probes for detecting an aneuploid of a Brassica oleracea plant, including at least one or more primers with the nucleotide sequences shown in SEQ ID NOs: 1 to 18; and at least one or more fluorescent dye modified probes with the nucleotide sequences shown in SEQ ID NOs: 19-27.

The aneuploid detection method of the present invention is used to estimate the frequency of chromosome aneuploid occurrence for each Brassica oleracea plant line. Using this method, the present invention can provide a method for crossing a culture of Brassica oleracea, including the selection of a line with a low incidence of aneuploids.

The aneuploid detection method of the present invention is used so that a method can be provided to control the quality of Brassica oleracea seeds, including testing the degree of aneuploid contamination contained in seeds from a Brassica oleracea seed lot.

The level of aneuploids contained in the seeds is assessed by a germination test. Such testing requires a large field and a growing period of several months. In addition, it was not possible to obtain accurate results unless germination was assessed by a qualified expert. By the seed quality control method of the present invention, aneuploid content test can be performed in a compact manner and fast and accurate results can be obtained.

The aneuploid detection method of the present invention is used so that a method can be provided for quality control of Brassica oleracea plants, including testing the degree of contamination with aneuploids contained in a batch of Brassica oleracea seeds.

According to the quality control method of the Brassica oleracea plant of the present invention, it is also possible to test newly emerged cotyledons from germinating seeds as material. Additionally, if the test for aneuploids is carried out before the planting stage, only normal individuals can be selected and cultured. As a result, ordinary fresh produce can be harvested from almost all plants.

[EXAMPLES]

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples. For example, a test sample from which DNA is isolated may be at any stage of growth. You can use seeds, cotyledons, true leaves, roots and any tissue. No special techniques are required for DNA isolation, provided that the DNA is purified to such an extent that PCR can be carried out without problems. As the fluorescent dye, any fluorescent dye can be used as long as it is a dye mainly used in the real-time PCR method.

As shown in the following examples, the method of the present invention is versatile and allows the detection of aneuploids in different crops such as broccoli, cauliflower and cabbage using a common marker. In addition, the present inventors confirmed in addition to these examples that this method can be carried out on a large number of lines, and this method is not limited to the line used in the following examples.

Example 1: Getting a token

PCR was performed using primers (10-mers) for randomly amplified polymorphic DNA (RAPD), Operon Technologies, Inc., and primers for sequence related amplified polymorphism (SRAP), developed by the present inventors, and DNA from the F2 generation of broccoli as matrices, and thus built a Brassica oleracea linkage map.

Then, the linkage map constructed by the authors of the present invention was compared with the article: I. Parkin et al., Genetics 171 (2005) p. 765, Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana, X. Cheng et al., Theoretical Applied Genetics 118 (2009) p. 1121, Development and genetic mapping of microsatellite markers from genome survey sequences in Brassica napus, and with public information registered with the NCBI, and thus analyzed associations between markers located in linkage groups and linkage maps in the public domain.

In addition, it was necessary to modify the markers on the chromosomes to expand the use to include all species of Brassica oleracea, and thus characteristic lines were selected from the broad genetic resources of cabbage, broccoli and cauliflower. DNA from these lines was used as templates to identify regions without SNPs and the primers were then redesigned.

These markers were used to perform PCR using nine types of trisomic plants as templates (FIG. 1) in which each of chromosomes 1 to 9 identified during marker development became trisomic. It was confirmed that there was no non-specific amplification from other chromosomes affecting the determination, and then each of the markers was considered as a marker specific to one of the chromosomes.

Further, hydrolysis probes labeled with three types of fluorescent dyes, including FAM, HEX, and Cy5, have been developed to perform a simpler and more stable multiplex fluorescence method. The goal was to design primers and probes so that even if three types of markers are mixed in the reaction solution (i.e. even if six types of primers and three types of probes are mixed), the markers do not interfere with the probes and reproducible results can be obtained. As a rule, when multiplex PCR is carried out, complex reactions occur, since the formation of primer dimers and annealing of another primer with an amplified DNA fragment occurs. Thus, it is difficult to construct a system that would consistently detect the difference between two and three chromosomes (1.5-fold difference) with respect to crude template DNA. However, the authors of the present invention have successfully developed the markers shown in tables 1 and 2, as a result of multiple improvements in the sequence of markers and intensive research.

Further, in order to conduct a more accurate and stable test, markers obtained from nine chromosomes were divided into three groups of three markers each (see Example 4, which will be described below).

[Table 1] List of PCR primer sequences

[Table 2] List of fluorescent probe sequences

FIG. 2 shows the basic principle associated with relative quantification using real-time PCR.

When the amount of DNA added as a template to the PCR reaction solution is constant, then, for example, in the case of trisomy on chromosome 6, the amplification curve of the marker located on chromosome 6 increases faster than in a normal individual. In the case of relative quantification, the intersection point between the amplification curve and the threshold line set according to a certain standard is determined as a Ct value and compared with the Ct value of each of the markers of other chromosomes, thereby achieving an estimate of the number of target chromosomes.

In FIG. 3 shows an example of the calculation result of the relative amount obtained by real-time PCR. In this test, multiplex PCR was performed using a fluorescent probe method using 96 individuals of F1 cultivars of broccoli as materials, a chromosome 4 marker and a chromosome 6 marker. and "Premix Ex Taq (Perfect Real Time)" by Takara Bio Inc. used as a reagent for the reaction. In the figure, the x-axis shows the number of individuals and the y-axis shows the relative amount of the chromosome 6 marker calculated by the ΔΔCt method using the chromosome 4 marker as a standard.

Among the 96 individuals tested, 88 normal type individuals showed a value of about 1, while 5 individuals with trisomy 6 showed a value of about 1.4, and 3 individuals with trisomy 4 showed a value of about 0.7. When the PCR amplification efficiency was assumed to be double per cycle, the theoretical values for trisomy 6 and trisomy 4 were 1.5 and 0.66, respectively, and thus the actual values were close to these values.

Example 2 Observation of a chromosome in a trisomic broccoli plant

Among the plants that were determined to be trisomic by real-time PCR, trisomy 6, trisomy 7, and trisomy 2 were used as materials, and chromosomes were observed.

Anthers were extracted from broccoli buds 1 to 2 mm long and stained with 1% orcein in acetic acid solution. Chromosomes were observed during primary metaphase (MI stage) of meiosis of pollen mother cells.

The results were as shown in FIG. four.

As shown in FIG. 4, in the case of a chromosome of a normal individual, nine bivalent chromosomes (2n=18) were observed. Meanwhile, in the case of a chromosome observed in a trisomic plant, nine bivalent chromosomes and one chromosome (indicated by an arrow) (2n+1) were observed.

Example 3 Detection of an atypical plant in broccoli

F1 seeds of 'SBR-48' broccoli cultivar developed by SAKATA SEED CORPORATION were sown in nursery trays. DNA from 445 individuals was extracted from newly emerged cotyledons from germinating seeds and real-time PCR was performed using the SYBR method using markers specific to each chromosome.

Specifically, primers with the nucleotide sequences shown in SEQ ID NO: 1 to 18 were used as chromosome-specific markers. SYBR Green I (purchased from Roche) was added to a normal PCR reaction solution as an intercalator resulting in a final concentration of 1/20000, followed by PCR. For real-time PCR, a LightCycler 480 System II from Roche was used. The method used for PCR included incubation at 95°C for 1 minute, followed by 40 cycles of three-step PCR at 95°C for 15 seconds, 60°C for 30 seconds and 72°C for 30 seconds. When measuring the signal with SYBR Green I, a filter with an excitation wavelength of 465 nm and a detection wavelength of 510 nm was used.

Based on the Ct value obtained by the second derivative method, the relative amount was calculated by the ΔΔCt method using a marker on chromosome 9 as a standard.

The results were as shown in Table 3.

These results are summarized in Table 3 for each chromosome and thus it is clear that aneuploids are included in the ratio as shown in Table 4.

In this example, trisomy 3 did not occur. However, as shown in FIG. 1, trisomy 3 appears in rare cases (the phenotypic characteristics of trisomy for each chromosome are the same as shown in FIG. 7). Note that the present inventors have separately confirmed that there are no problems with the developed markers, including trisomy 3.

Table 3. Aneuploid testing result in F1 broccoli plants (raw data obtained by PCR based on the SYBR method (value calculated using the ΔΔCt method))

[Table 3-1]

SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR C1 C2 C3 C4 C5 C6 C7 C8 C9 С1/С9 С2/С9 С3/С9 С4/С9 С5/С9 С6/С9 С7/С9 С8/С9 С9/С9 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 1 0.98 1.06 1.04 1.11 0.95 1.08 0.94 0.96 1.00 Normal 2 1.03 0.98 0.99 1.02 0.98 1.04 0.95 0.91 1.00 Normal 3 0.86 0.96 1.13 1.23 1.04 0.92 1.10 1.18 1.00 Aneuploid (+C4) four 0.98 1.03 0.97 0.99 1.03 1.03 0.95 0.98 1.00 Normal five 1.10 1.00 0.94 1.12 0.93 1.07 0.95 0.99 1.00 Normal 6 0.96 0.99 0.95 0.94 0.96 1.03 0.95 0.95 1.00 Normal 7 1.01 1.05 1.01 1.02 1.00 1.05 0.93 0.96 1.00 Normal 8 1.03 0.99 0.98 1.06 1.00 1.07 0.95 0.94 1.00 Normal nine 1.03 0.96 0.94 1.02 0.96 1.01 0.95 0.95 1.00 Normal 10 1.03 0.93 1.01 0.99 1.01 0.92 1.00 0.98 1.00 Normal eleven 0.95 0.98 0.96 0.96 0.94 1.05 0.92 0.90 1.00 Normal 12 1.03 1.01 0.93 1.06 0.94 1.08 0.94 1.09 1.00 Normal 13 1.05 1.05 1.02 1.01 1.04 1.08 0.97 1.10 1.00 Normal

fourteen 1.01 1.04 1.08 1.00 1.03 1.08 0.95 0.96 1.00 Normal 15 1.08 1.08 1.07 1.01 1.08 1.06 0.96 0.96 1.00 Normal sixteen 1.00 1.02 0.98 1.02 1.00 1.07 0.98 0.95 1.00 Normal 17 0.97 1.02 0.99 1.01 0.96 1.05 0.98 0.98 1.00 Normal eighteen 1.02 1.03 1.04 1.12 0.99 1.05 0.92 1.00 1.00 Normal nineteen 1.03 0.98 0.99 1.14 1.00 1.02 0.93 0.92 1.00 Normal 20 1.03 1.04 0.98 1.12 1.03 1.03 0.97 1.02 1.00 Normal 21 1.04 1.06 0.99 1.04 1.03 1.08 0.99 1.03 1.00 Normal 22 1.00 0.94 0.98 1.02 0.99 1.00 1.00 1.01 1.00 Normal 23 0.96 0.97 0.97 1.03 0.98 1.02 0.97 1.02 1.00 Normal 24 0.98 1.03 1.01 0.96 1.01 1.05 0.98 0.98 1.00 Normal 25 0.91 1.00 1.06 1.00 1.00 1.02 0.96 1.00 1.00 Normal 26 0.92 1.00 1.01 1.02 1.03 1.06 1.00 0.99 1.00 Normal 27 0.99 0.98 0.91 1.03 0.98 0.99 0.92 0.98 1.00 Normal 28 0.97 1.01 0.94 1.01 1.00 1.04 0.92 0.95 1.00 Normal 29 1.04 1.03 0.98 1.10 1.02 1.10 0.95 1.02 1.00 Normal thirty 1.00 1.02 1.01 1.05 1.07 1.08 1.02 1.00 1.00 Normal 31 0.97 0.97 0.99 0.97 J.D2 0.98 0.95 0.96 1.00 Normal 32 0.90 0.97 0.97 0.98 0.99 1.00 1.41 0.92 1.00 Aneuploid (+C7)

33 0.59 0.90 0.91 0.83 0.92 0.89 ].25 0.90 1.00 Aneuploid (other) 34 1.01 0.99 0.99 0.99 1.03 1.04 0.93 0.96 1.00 Normal 35 0.95 1.04 1.01 0.98 1.07 0.95 1.09 1.02 1.00 Normal 36 1.43 1.00 0.97 1.05 1.02 1.03 0.96 0.98 1.00 Aneuploid (+C1) 37 0.98 1.00 0.95 0.99 1.05 1.06 0.97 0.91 1.00 Normal 38 1.03 1.02 1.01 1.01 0.99 1.08 0.92 0.95 1.00 Normal 39 1.01 1.00 J.01 1.06 J.00 1.04 0.95 0.99 J.00 Normal 40 1.02 1.02 0.92 1.08 0.99 1.09 0.92 0.98 1.00 Normal 41 0.99 1.02 0.95 1.02 0.98 0.98 0.94 0.92 1.00 Normal 42 1.03 1.05 1.10 1.12 1.12 1.15 1.04 1.02 1.00 Normal 43 1.03 0.98 1.01 1.02 1.03 1.03 0.95 0.94 1.00 Normal 44 1.02 1.03 1.03 1.00 1.05 0.98 1.04 1.05 1.00 Normal 45 0.99 0.98 0.99 0.94 1.00 0.98 0.94 0.90 1.00 Normal 46 1.11 1.11 1.10 1.05 1.03 1.06 1.02 1.05 1.00 Normal 47 0.99 1.02 1.00 1.07 1.03 1.01 J.06 1.07 1.00 Normal 48 0.98 0.97 0.94 1.05 0.99 1.05 0.97 1.00 1.00 Normal 49 1.00 1.00 0.97 0.98 1.01 1.05 0.93 0.91 1.00 Normal

50 1.12 1.10 1.01 1.09 0.93 1.14 0.93 1.00 1.00 Normal 51 1.01 1.02 0.99 1.02 1.00 1.12 0.95 1.00 1.00 Normal 52 1.01 1.00 0.94 1.01 1.01 1.00 0.94 0.94 1.00 Normal 53 1.03 1.05 1.04 1.05 1.04 1.03 1.00 1.00 1.00 Normal 54 0.99 1.03 1.01 0.99 1.00 1.05 0.97 0.96 1.00 Normal 55 1.03 0.98 1.00 1.00 1.03 1.00 1.00 0.94 1.00 Normal 56 0.99 1.02 1.04 1.06 1.03 0.99 1.07 1.05 1.00 Normal 57 1.02 0.99 0.99 0.97 1.02 1.08 0.97 0.93 1.00 Normal 58 1.00 0.98 0.94 0.96 1.02 0.98 0.97 1.01 1.00 Normal 59 0.95 1.11 1.11 1.06 1.05 1.12 1.14 1.08 1.00 Normal 60 1.05 1.02 0.99 1.02 1.05 1.03 1.06 0.97 1.00 Normal 61 1.02 1.05 0.97 0.94 1.03 1.05 1.00 0.97 1.00 Normal 62 1.03 0.98 0.93 1.02 1.05 1.11 0.95 0.94 1.00 Normal 63 1.05 1.02 0.97 1.08 1.03 1.05 0.95 0.96 1.00 Normal 64 1.08 0.96 0.94 1.10 0.98 1.10 0.93 1.00 1.00 Normal

[Table 3-2]

SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR C1 C2 C3 C4 C5 C6 C7 C8 C9 С1/С9 С2/С9 С3/С9 С4/С9 С5/С9 С6/С9 С7/С9 С8/С9 С9/С9

individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 65 1.00 0.98 0.0.98 1.12 1.02 1.03 0.95 1.04 1.00 Normal 66 0.92 1.00 1.07 1.10 1.08 0.93 1.07 1.07 1.00 Normal 67 0.95 1.02 1.05 1.00 1.05 0.91 1.08 1.00 1.00 Normal 68 0.98 1.00 0.99 0.95 0.98 0.91 0.97 1.01 1.00 Normal 69 0.99 0.46 1.14 1.11 1.13 0.94 0.23 1.10 1.00 Aneuploid (others) 70 0.99 0.97 0.95 1.02 1.00 1.03 0.94 0.98 1.00 Normal 71 1.09 1.10 0.95 1.05 1.03 1.11 0.97 1.02 1.00 Normal 72 1.09 1.04 1.01 1.07 1.12 1.07 0.99 1.04 1.00 Normal 73 1.04 0.96 0.95 1.05 1.03 1.07 0.97 1.00 1.00 Normal 74 1.00 1.05 1.03 1.09 1.10 1.08 1.04 1.10 1.00 Normal

75 0.99 1.01 0.93 1.11 1.00 1.01 1.49 1.01 1.00 Aneuploid (+C7) 76 1.02 1.00 0.97 1.02 0.98 1.02 0.99 1.01 1.00 Normal 77 1.02 1.01 0.99 1.03 0.99 1.00 1.01 0.99 1.00 Normal 78 1.01 0.98 0.99 1.02 1.03 1.03 1.02 0.97 1.00 Normal 79 1.03 1.03 0.98 1.02 1.00 1.03 0.98 0.96 1.00 Normal 80 0.91 1.01 1.05 1.02 1.04 0.92 1.07 1.05 1.00 Normal 81 0.92 1.00 1.02 0.97 1.08 1.60 1.07 0.97 1.00 Aneuploid (+C6) 82 0.96 0.99 0.99 1.00 1.06 0.97 1.08 1.08 1.00 Normal 83 1.05 1.00 0.97 1.00 1.01 1.00 0.99 1.00 1.00 Normal 84 1.03 1.03 1.01 1.04 1.10 1.05 1.00 0.97 1.00 Normal 85 0.61 0.65 0.66 0.65 0.67 0.64 0.67 0.68 1.00 Aneuploid (+C9)

86 0.96 1.01 0.96 1.06 1.00 1.00 0.98 0.93 1.00 Normal 87 1.03 1.01 0.94 1.05 1.01 1.04 0.97 1.00 1.00 Normal 88 1.00 0.95 0.95 1.01 1.00 1.02 0.95 1.04 1.00 Normal 89 0.93 0.92 0.95 1.08 0.94 0.92 0.93 1.02 1.00 Normal 90 1.00 0.98 1.04 1.01 1.00 1.04 1.00 1.05 1.00 Normal 91 1.11 1.12 1.13 1.12 1.14 1.13 1.10 1.12 1.00 Normal 92 1.01 1.00 0.95 1.02 0.97 1.00 0.94 1.00 1.00 Normal 93 1.02 0.98 0.96 0.98 0.99 1.03 0.97 0.96 1.00 Normal 94 0.96
0.99 1.01 0.94 0.99 1.00 0.97 0.98 1.00 Normal 95 1.00 1.01 0.98 1.00 0.98 0.99 0.96 1.05 1.00 Normal 96 1.04 1.07 1.09 0.99 0.99 1.05 0.98 1.00 1.00 Normal

97 1.03 1.00 1.07 1.05 1.08 1.06 1.02 1.00 1.00 Normal 98 1.07 1.14 1.07 1.13 1.05 1.15 1.00 1.05 1.00 Normal 99 1.03 0.98 0.99 1.01 0.98 0.94 0.97 1.00 1.00 Normal one hundred 0.96 0.97 1.10 1.08 1.06 0.93 1.11 1.09 1.00 Normal 101 0.97 0.98 1.01 0.93 1.03 1.03 1.02 0.96 1.00 Normal 102 1.03 0.96 1.02 0.96 1.00 0.95 0.96 1.03 1.00 Normal 103 1.04 1.00 1.05 0.99 1.01 1.03 1.02 0.98 1.00 Normal 104 1.08 1.07 1.01 0.99 1.03 1.02 0.97 0.96 1.00 Normal 105 1.02 1.01 0.98 0.94 0399 0.98 0.95 0.96 1.00 Normal 106 0.96 1.00 1.05 1.02 1.07 0.95 1.66 1.04 1.00 Aneuploid (+C7) 107 1.02 1.00 0.96 1.04 1.00 1.00 0.95 1.00 1.00 Normal

108 1.05 1.05 1.01 0.98 1.00 0.98 1.02 0.98 1.00 Normal 109 0.99 1.00 0.99 0.98 1.00 1.00 0.94 0.94 1.00 Normal 110 1.00 1.02 1.07 1.02 1.06 1.03 0.98 1.01 1.00 Normal 111 1.05 1.05 1.01 1.11 1.07 1.05 1.00 1.02 1.00 Normal 112 1.06 1.00 1.00 0.94 0.96 0.98 0.97 1.01 1.00 Normal 113 1.03 1.04 1.02 0.94 1.00 0.96 1.02 1.00 1.00 Normal 114 1.05 1.00 0.99 0.94 1.02 1.04 0.95 0.91 1.00 Normal 115 1.01 0.96 0.93 1.00 0.96 1.05 0.93 0.99 1.00 Normal 116 1.05 1.00 1.02 1.04 1.02 1.02 1.00 1.06 1.00 Normal 117 1.02 1.01 0.99 1.00 0.99 .098 1.00 1.04 1.00 Normal 118 0.98 1.01 1.01 1.01 0.99 0.96 0.93 0.98 1.00 Normal

119 1.06 1.00 0.99 1.02 1.02 1.02 0.97 1.05 1.00 Normal 120 0.99 1.01 1.00 0.99 1.00 1.04 0.95 0.93 1.00 Normal 121 1.04 1.02 1.04 1.14 1.00 1.12 1.00 1.02 1.00 Normal 122 1.03 0.96 0.98 0.94 0.98 0.96 1.04 0.89 1.00 Normal 123 1.01 1.02 1.00 1.02 1.05 1.04 1.00 1.00 1.00 Normal 124 1.00 1.00 1.01 0.97 1.00 1.00 0.95 0.96 1.00 Normal 125 0.99 0.98 1.05 0.99 1.00 1.00 0.97 1.02 1.00 Normal 126 0.97 0.95 1.07 0.96 0.98 0.96 0.94 1.00 1.00 Normal 127 1.09 1.05 1.00 0.96 1.00 1.04 1.02 0.97 1.00 Normal 128 1.00 1.00 1.00 0.99 0.98 1.00 0.94 0.98 1.00 Normal 129 1.00 1.02 0.99 0.94 1.00 1.00 0.97 0.98 1.00 Normal 130 0.98 1.03 1.01 0.99 1.01 1.03 1.00 0.96 1.00 Normal

131 0.98 1.08 1.07 1.11 1.08 1.06 1.02 1.04 1.00 Normal

[Table 3-3]

SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR C1 C2 C3 C4 C5 C6 C7 C8 C9 С1/С9 С2/С9 С3/С9 С4/С9 С5/С9 С6/С9 С7/С9 С8/С9 С9/С9 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 132 1.00 1.00 0.95 1.06 1.00 1.01 0.97 0.92 1.00 Normal 133 1.04 1.02 1.01 1.14 1.07 1.00 1.O1 1.03 1.00 Normal 134 1.03 0.96 0.95 0.99 1.01 0.96 1.02 0.97 1.00 Normal 135 1.11 1.03 1.05 1.07 1.07 1.10 1.05 1.05 1.00 Normal 136 1.03 1.00 0.99 0.97 0.99 0.97 0.99 1.00 1.00 Normal 137 1.07 1.04 1.02 1.07 1.04 1.01 1.02 1.02 1.00 Normal 138 1.08 1.00 1.05 0.98 1.00 0.98 1.02 1.02 1.00 Normal 139 1.04 1.07 1.07 1.01 0.99 0.98 1.05 1.04 1.00 Normal 140 1.06 1.07 1.06 1.01 1.02 1.04 1.06 1.07 1.00 Normal 141 1.09 1.07 1.01 1.05 1.08 1.08 1.04 1.01 1.00 Normal 142 1.06 1.09 1.06 1.11 1.06 1.08 1.05 1.02 1.00 Normal 143 1.03 1.08 1.04 1.04 1.04 1.03 1.04 0.98 1.00 Normal 144 1.04 1.03 1.04 1.00 1.03 0.97 1.06 0.99 1.00 Normal

145 1.07 1.11 1.06 1.02 1.10 1.05 1.07 1.01 1.00 Normal 146 1.09 0.51 1.09 1.05 1.03 1.03 1.07 1.03 1.00 Aneuploid (others) 147 1.03 0.97 0.97 1.03 0.96 0.99 0.95 1.00 1.00 Normal 148 1.14 1.14 1.09 1.08 1.12 1.08 1.10 1.07 1.00 Normal 149 0.95 0.96 1.04 0.99 1.05 0.99 1.05 1.02 1.00 Normal 150 0.98 1.01 1.01 1.02 1.06 1.00 1.05 1.1 1.00 Normal 151 1.05 1.02 1.00 1.08 1.03 1.13 0.97 1.04 1.00 Normal 152 0.96 1.02 1.05 1.02 1.04 0.98 1.50 1.02 1.00 Aneuploid (+C7) 153 1.03 1.00 0.93 1.08 0.98 1.06 0.92 0.99 1.00 Normal 154 0.93 1.00 1.01 1.03 1.07 0.93 1.07 1.02 1.00 Normal 155 0.94 1.00 1.01 0.98 0.96 1.00 0.97 0.98 1.00 Normal 156 1.01 1.01 1.04 1.04 1.01 0.94 1.01 1.00 1.00 Normal 157 1.02 1.00 0.98 0.96 0.97 0.98 0.95 0.97 1.00 Normal 158 1.11 1.11 1.13 1.0R 1.05 1.60 1.11 1.10 1.00 Aneuploid (+C6) 159 0.98 1.02 0.93 1.00 0.95 0.99 0.95 0.94 1.00 Normal 160 1.00 0.99 1.00 0.96 1.00 0.91 0.99 0.97 1.00 Normal 161 1.00 1.02 0.97 0.98 1.37 1.60 0.92 1.00 1.00 Aneuploid (others) 162 0.97 1.06 1.02 1.05 0.96 1.04 1.04 1.04 1.00 Normal 163 1.06 1.05 0.97 1.05 1.00 1.07 0.99 0.98 1.00 Normal 64 1.03 1.11 1.04 1.03 0.98 1.03 1.07 1.02 1.00 Normal 165 0.97 1.01 1.08 1.05 1.02 0.91 1.09 1.00 1.00 Normal

166 0.98 0.97 0.93 0.93 0.93 0.96 0.93 0.92 1.00 Normal 167 1.11 1.13 1.07 1.04 0.90 1.04 0.98 1.02 1.00 Normal 168 1.11 1.11 1.04 1.08 0.87 1.07 1.00 1.00 1.00 Normal 169 0.96 0.96 1.01 0.87 0.92 1.00 0.97 1.00 1.00 Normal 170 0.94 1.11 1.04 0.89 0.90 0.91 1.01 0.65 1.00 Normal 171 0.98 0.9B 0.97 1.00 0.96 1.06 1.01 0.07 1.00 Normal 172 1.06 1.10 1.09 1.10 1.09 1.11 1.05 1.05 1.00 Normal 173 1.00 0.97 1.00 1.04 1.05 1.10 0.85 0.95 1.00 Normal 174 1.00 1.05 0.95 1.14 0.99 0.98 1.01 1.05 1.00 Normal 175 0.98 0.96 0.99 0.99 0.96 0.96 0.95 0.93 1.00 Normal 176 1.00 0.98 0.99 0.97 1.00 1.02 0.98 0.95 1.00 Normal 177 1.05 1.03 1.01 1.00 1.06 1.07 1.04 1.06 1.00 Normal 178 1.03 1.02 1.03 0.98 0.89 0.98 0.98 0.98 1.00 Normal 179 0.98 0.96 1.01 0.91 0.97 0.96 0.95 1.04 1.00 Normal 1B0 1.02 1.00 1.01 0.93 1.00 1.00 0.98 0.97 1.00 Normal 181 1.05 1.07 1.03 1.02 0.96 1.08 0.95 1.00 1.00 Normal 82 1.01 1.01 1.01 0.94 0.97 0.96 1.00 0.96 1.00 Normal 183 1.07 1.02 1.04 1.00 1.00 1.03 1.01 0.99 1.00 Normal 184 0.98 1.04 0.99 1.02 0.96 1.08 0.95 0.96 1.00 Normal 185 1.08 1.11 1.06 1.06 1.02 1.11 1.00 1.04 1.00 Normal 186 0.98 1.07 1.05 1.06 0.99 1.06 1.01 1.07 1.00 Normal 187 1.00 1.03 1.03 1.00 1.03 1.06 1.04 1.00 1.00 Normal

188 1.03 1.04 1.01 1.07 1.00 1.01 J.05 1.06 1.00 Normal 189 1.06 0.98 1.00 0.96 0.98 0.92 1.06 0.96 1.00 Normal 190 1.11 1.08 1.09 1.00 0.98 1.05 1.06 1.05 1.00 Normal 191 1.02 1.05 1.04 0.96 1.03 1.05 1.00 1.05 1.00 Normal 192 0.99 1.01 1.04 1.00 1.00 0.97 0.97 1.06 1.00 Normal 193 1.10 1.07 1.02 0.96 0.99 1.03 1.02 1.00 1.00 Normal 194 1.03 1.00 1.04 0.99 0.94 1.01 1.07 1.02 1.00 Normal 195 1.03 1.54 1.01 0.93 0.89 1.03 1.02 0.96 1.00 Aneuploid (+C2) 196 0.98 1.00 0.97 0.99 0.99 0.99 1.00 0.93 1.00 Normal 197 1.00 1.04 0.95 1.00 1.00 1.00 0.96 0.94 1.00 Normal 198 1.04 1.05 1.04 1.04 1.02 1.11 0.99 1.03 1.00 Normal

[Table 3-4]

SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR C1 C2 C3 C4 C5 C6 C7 C8 C9 С1/С9 С2/С9 С3/С9 С4/С9 С5/С9 С6/С9 С7/С9 С8/С9 С9/С9 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 199 1.01 1.02 0.95 0.44 0.89 0.98 0.93 0.90 1.00 Aneuploid (others) 200 1.02 0.99 0.97 0.91 0.95 0.95 0.99 0.99 1.00 Normal 201 0.94 0.95 0.97 0.91 0.96 0.91 0.90 0.92 1.00 Normal 202 1.06 1.06 1.01 1.03 1.03 1.05 1.02 1.00 1.00 Normal 203 1.02 1.05 1.01 0.92 0.98 0.99 1.01 1.00 1.00 Normal 204 1.04 1.05 1.05 0.98 0.97 1.00 1.02 0.96 1.00 Normal

205 1.03 1.00 0.95 1.00 1.03 1.05 0.97 1.00 1.00 Normal 206 1.04 1.05 0.99 0.98 1.05 1.03 1.06 1.00 1.00 Normal 207 0.98 1.02 0.97 0.96 0.96 0.97 0.97 0.92 1.00 Normal 208 0.98 1.11 0.99 1.03 1.01 0.97 0.95 1.06 1.00 Normal 209 1.05 1.03 1.00 0.98 1.01 1.00 1.01 0.94 1.00 Normal 210 1.00 1.05 1.07 0.92 0.91 0.98 1.00 1.04 1.00 Normal 211 1.11 1.08 1.12 1.03 1.06 1.01 1.11 1.09 1.00 Normal 212 1.00 1.02 0.99 0.96 1.01 0.96 0.97 0.95 1.00 Normal 213 0.98 1.05 1.01 0.96 1.03 1.01 1.04 0.98 1.00 Normal 214 1.05 1.09 0.99 0.99 1.02 1.00 1.00 0.96 1.00 Normal 215 1.00 0.98 0.97 1.01 1.02 0.96 0.98 1.02 1.00 Normal 216 0.98 1.04 0.97 0.96 1.00 Q.98 0.96 1.03 1.00 Normal 217 0.98 1.02 1.00 1.02 1.03 0.94 1.02 0.97 1.00 Normal 218 1.03 1.00 1.00 1.02 1.01 0.95 1.00 1.08 1.00 Normal 219 1.13 0.98 0.97 0.99 0.97 0.96 0.95 1.04 1.00 Normal 220 1.01 0.97 0.98 0.92 0.94 0.96 0.93 0.91 1.00 Normal 221 1.11 1.10 1.06 1.03 1.08 1.06 0.98 1.05 1.00 Normal 222 0.98 1.00 1.00 1.00 1.05 0.98 0.95 1.04 1.00 Normal 223 1.03 0.96 0.95 1.05 0.96 0.91 0.97 1.05 1.00 Normal 224 0.97 1.06 0.99 1.02 1.00 1.03 0.97 1.05 1.00 Normal 225 1.03 1.02 .04 1.11 1.04 1.05 0.94 1.01 1.00 Normal 226 0.98 1.03 0.95 1.00 1.03 1.02 1.02 1.00 1.00 Normal

227 0.99 0.94 0.96 1.03 0.97 0.98 0.97 1.02 1.00 Normal 228 1.00 0.97 0.99 1.08 0.99 0.90 1.01 1.01 1.00 Normal 229 1.04 0.97 0.99 0.98 0.98 0.94 1.00 1.00 1.00 Normal 230 1.03 1.05 1.10 1.04 1.01 0.98 1.04 1.03 1.00 Normal 231 1.05 1.02 1.04 1.00 0.97 0.98 1.00 1.02 1.00 Normal 232 1.00 0.97 0.95 0.93 0.96 0.94 0.97 1.03 1.00 Normal 233 1.05 1.06 1.04 0.97 1.05 1.05 1.00 0.96 1.00 Normal 234 1.13 1.08 0.99 1.11 1.02 1.00 0.96 1.02 1.00 Normal 235 0.94 0.99 1.04 0.99 1.03 0.99 0.99 0.98 1.00 Normal 236 0.99 1.02 1.01 0.97 1.03 1.00 1.00 0.97 1.00 Normal 237 1.07 1.03 1.00 1.06 1.00 1.10 0.83 0.97 1.00 Normal 238 1.03 1.00 1.01 1.00 1.01 1.00 0.99 0.99 1.00 Normal 239 1.06 1.01 1.02 1.01 0.99 1.00 1.00 1.00 1.00 Normal 240 1.03 1.02 1.04 1.00 0.91 0.96 1.02 1.00 1.00 Normal 24a 1.10 1.04 1.05 1.02 0.97 1.03 1.01 1.02 1.00 Normal 242 1.05 1.08 1.12 1.00 1.00 1.10 1.02 1.07 1.00 Normal 243 0.98 0.95 0.99 0.92 0.94 0.95 0.98 0.94 1.00 Normal 244 1.05 1.03 1.04 0.98 1.05 0.98 1.04 1.00 1.00 Normal 245 1.00 1.04 1.07 1.02 1.11 1.40 1.09 1.05 1.00 aneuploid (+C6)

246 0.98 0.98 1.01 1.00 1.00 0.97 0.97 0.94 1.00 Normal 247 1.00 1.00 1.04 1.01 1.06 1.00 1.03 0.97 1.00 Normal 248 1.00 1.02 0.99 0.96 1.00 1.02 0.97 1.02 1.00 Normal 249 0.95 0.92 0.94 0.97 0.95 0.96 0.95 0.91 1.00 Normal 250 0.96 0.99 1.06 1.05 1.05 0.90 1.07 1.02 1.00 Normal 251 1.03 0.94 1.02 1.00 1.03 1.03 1.00 1.03 1.00 Normal 252 0.96 0.96 0.98 0.96 J.00 1.00 0.95 1.02 1.00 Normal 253 0.98 1.02 1.04 0.96 1.00 0.98 0.96 0.92 1.00 Normal 254 1.05 1.11 1.04 1.00 1.05 1.03 1.04 0.98 1.00 Normal 255 1.08 1.03 1.05 1.09 1.02 1.05 1.00 1.06 1.00 Normal 256 0.98 1.05 1.03 1.02 1.02 1.05 1.03 0.94 1.00 Normal 257 1.01 0.97 0.99 0.98 1.02 1.00 0.97 0.96 1.00 Normal 258 0.97 1.00 0.95 1.00 1.05 1.02 1.04 0.96 1.00 Normal 259 1.00 0.98 0.97 1.IZ 0.98 1.03 0.95 1.03 1.00 Normal 260 1.01 0.98 0.99 0.98 0.98 1.05 1.00 0.99 1.00 Normal 261 0.98 0.99 0.99 0.94 1.00 0.94 1.01 1.08 1.00 Normal 262 0.96 1.01 0.95 0.96 0.99 0.94 0.99 0.95 1.00 Normal 263 1.01 0.96 0.91 0.92 0.94 0.96 0.93 0.92 1.00 Normal 264 1.05 1.03 1.01 0.98 0.99 0.96 1.06 0.98 1.00 Normal 265 0.98 1.07 0.99 1.05 0.99 1.05 1.01 1.08 1.00 Normal

[Table 3-5]

SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR

C1 C2 C3 C4 C5 C6 C7 C8 C9 С1/С9 С2/С9 С3/С9 С4/С9 С5/С9 С6/С9 С7/С9 С8/С9 С9/С9 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 266 0.98 1.00 0.95 0.97 0.96 0.98 0.97 0.97 1.00 Normal 267 1.02 1.07 1.05 1.02 1.00 1.04 1.04 1.02 1.00 Normal 268 0.91 0.99 0.95 1.00 0.97 0.98 1.04 1.02 1.00 Normal 269 0.82 1.51 0.95 1.00 0.96 0.91 0.95 0.98 1.00 aneuploid (+C2) 270 1.09 1.11 1.06 1.11 1.12 1.13 1.09 1.09 1.00 Normal 271 0.99 1.01 0.99 1.04 1.00 1.00 1.02 1.02 1.00 Normal 272 0.99 0.97 0.97 1.01 1.01 1.54 0.97 1.05 1.00 aneuploid (+C6) 273 1.01 0.98 0.93 0.92 0.98 1.00 0.93 1.02 1.00 Normal 274 0.94 0.95 0.93 0.85 0.90 0.93 0.95 0.89 1.00 Normal 275 0.99 0.95 0.96 0.89 0.91 0.94 0.96 0.98 1.00 Normal 276 0.96 0.98 0.95 1.00 1.00 0.96 0.95 1.04 1.00 Normal 277 0.97 1.00 0.98 0.93 0.98 0.95 1.00 1.03 1.00 Normal 278 0.93 0.96 1.02 0.94 0.98 0.96 0.97 0.98 1.00 Normal 279 0.96 0.99 0.98 0.93 1.00 0.98 0.95 0.94 1.00 Normal 280 0.96 1.02 1.02 1.00 1.00 0.98 1.08 1.08 1.00 Normal 281 1.00 0.95 0.93 0.96 0.97 0.98 0.97 0.94 1.00 Normal 282 0.96 0.99 0.97 1.00 1.00 0.98 1.02 1.04 1.00 Normal

283 0.99 0.91 0.90 0.96 0.93 0.92 0.89 0.94 1.00 Normal 284 0.96 0.90 0.92 0.98 0.87 0.97 0.87 0.95 1.00 Normal 285 0.96 0.94 0.95 0.93 0.99 0.96 0.96 0.94 1.00 Normal 286 1.03 0.96 0.97 0.94 1.01 0.96 1.02 0.98 1.00 Normal 287 0.92 0.98 0.95 0.96 0.98 0.96 1.02 1.08 1.00 Normal 288 0.92 0.98 1.10 0.97 0.96 1.00 0.97 1.04 1.00 Normal 289 1.00 1.04 0.99 0.96 1.01 0.96 1.03 0.97 1.00 Normal 290 0.97 1.01 0.97 0.99 1.00 1.02 1.00 1.00 1.00 Normal 291 1.02 1.01 1.01 0.96 0.98 1.02 1.03 1.05 1.00 Normal 292 0.90 1.01 0.93 0.95 0.98 0.92 1.04 1.00 1.00 Normal 293 0.94 0.99 0.93 0.99 0.96 0.99 0.97 0.94 1.00 Normal 294 0.96 1.00 0.99 1.00 0.99 0.96 1.02 0.96 1.00 Normal 295 0.98 0.92 0.98 0.93 0.97 0.93 0.95 0.92 1.00 Normal 296 0.92 0.98 0.94 0.92 0.96 0.96 0.99 0.93 1.00 Normal 297 0.91 1.00 1.04 0.93 1.03 0.91 1.07 1.04 1.00 Normal 298 0.94 1.05 0.97 1.06 0.98 0.95 0.95 1.00 1.00 Normal 299 0.98 0.98 0.93 0.94 0.94 0.91 0.95 0.98 1.00 Normal 300 0.90 0.95 0.97 0.98 0.99 0.96 1.05 1.05 1.00 Normal 301 0.98 0.99 0.99 0.93 0.96 0.93 0.99 0.9d 1.00 Normal 302 0.96 0.96 0.98 0.96 1.00 1.01 1.01 0.98 1.00 Normal

303 0.95 0.98 1.00 0.92 0.96 0.96 1.00 0.94 1.00 Normal 304 0.99 1.00 0.93 0.96 0.96 0.98 0.99 0.97 1.00 Normal 305 0.96 0.96 1.00 1.05 0.98 1.05 0.95 0.99 1.00 Normal 306 0.92 0.92 0.95 0.92 0.93 0.89 0.94 0.94 1.00 Normal 307 1.00 1.05 0.99 1.02 0.96 1.03 0.99 1.05 1.00 Normal 308 0.89 0.91 0.93 0.91 0.98 0.89 0.96 1.05 1.00 Normal 309 0.92 0.99 1.00 0.94 0.98 0.96 1.05 1.04 1.00 Normal 310 0.58 0.60 0.60 0.57 0.59 0.57 0.59 0.63 1.00 aneuploid (+C9) 311 0.95 0.94 0.91 0.89 0.93 0.93 0.95 0.94 1.00 Normal 312 0.95 0.97 1.02 0.92 0.93 0.96 1.00 0.98 1.00 Normal 313 0.92 0.94 1.04 1.01 0.98 0.93 1.01 1.01 1.00 Normal 314 0.95 1.01 1.01 0.98 0.99 0.96 1.05 1.08 1.00 Normal 315 1.02 1.00 1.00 0.99 1.00 0.96 1.04 1.03 1.00 Normal 316 0.95 0.94 0.97 1.00 1.00 1.00 1.01 1.08 1.00 Normal 317 0.98 0.98 0.97 1.02 1.00 1.05 0.92 1.08 1.00 Normal 318 0.96 0.99 1.01 0.95 0.97 0.93 1.03 1.02 1.00 Normal 319 0.95 0.94 0.94 0.92 0.95 0.94 0.96 0.06 J.00 Normal 320 1.01 0.96 0.96 0.92 0.95 0.94 1.01 0.97 1.00 Normal 321 0.92 0.90 0.97 0.85 0.95 0.89 1.02 0.96 1.00 Normal 322 0.99 0.95 0.97 1.05 0.98 0.99 1.00 1.06 1.00 Normal 323 1.03 1.06 1.01 0.93 0.96 1.02 0.97 1.02 1.00 Normal

324 1.00 1.00 0.96 0.92 1.01 0.98 1.03 0.99 1.00 Normal 325 1.03 1.07 1.07 1.03 1.00 0.92 1.12 1.05 J.00 Normal 326 0.90 1.02 0.99 0.98 1.00 1.03 1.01 1.02 1.00 Normal 327 0.98 1.04 1.01 1.06 0.97 1.09 1.04 1.04 1.00 Normal 328 0.99 1.01 0.95 1.12 1.03 1.12 0.93 1.05 1.00 Normal 329 0.98 0.64 0.97 0.89 0.87 0.87 0.95 0.98 1.00 Normal 330 0.99 1.02 0.99 0.97 0.98 0.89 1.09 1.04 1.00 Normal 331 1.00 1.00 0.98 0.98 0.93 0.98 0.95 1.02 1.00 Normal 332 1.00 0.97 0.97 0.94 1.02 0.96 1.07 1.00 1.00 Normal

[Table 3-6]

SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR C1 C2 C3 C4 C5 C6 C7 C8 C9 С1/С9 С2/С9 С3/С9 С4/С9 С5/С9 С6/С9 С7/С9 С8/С9 С9/С9 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 333 1.03 1.01 1.02 1.05 0.92 1.02 0.92 1.01 1.00 Normal 334 0.96 1.00 0.99 1.02 0.99 0.96 1.00 1.10 1.00 Normal 335 0.97 1.11 1.12 1.01 0.97 1.03 1.01 1.11 1.00 Normal 336 1.00 1.01 1.01 1.12 1.01 1.05 1.02 1.08 1.00 Normal 337 1.04 0.98 0.99 1.05 0.96 1.05 1.00 0.98 1.00 Normal 338 0.92 0.94 1.01 1.00 1.00 0.95 1.00 1.03 1.Q0 Normal 339 1.01 0.96 1.01 1.06 0.98 0.95 1.03 1.10 1.00 Normal 340 0.98 0.97 0.94 1.02 0.92 1.05 1.01 0.98 1.00 Normal

341 0.92 0.97 0.93 0.90 0.94 0.92 1.00 1.00 1.00 Normal 342 1.06 1.03 1.06 1.02 0.99 0.96 1.07 1.07 1.00 Normal 343 0.96 0.94 0.97 0.96 1.00 0.94 1.00 1.02 1.00 Normal 344 0.99 d.98 0.97 0.97 1.03 0.95 0.97 0.99 1.00 Normal 345 0.94 0.99 0.99 1.06 0.96 0.95 0.93 1.00 1.00 Normal 346 0.92 1.00 0.96 1.07 0.93 1.03 1.02 1.02 1.00 Normal 347 1.03 1.00 0.93 1.11 0.97 1.02 0.96 0.96 1.00 Normal 348 0.97 0.96 0.99 0.97 0.96 1.09 0.62 0.89 1.00 Normal 349 1.05 0.99 0.97 1.05 1.01 10 1.02 0.95 1.00 Normal 350 1.01 1.03 1.01 1.06 1.02 0.99 0.9B 0.97 1.00 Normal 351 0.93 0.92 1.01 0.99 0.98 1.01 0.95 1.03 1.00 Normal 352 1.03 1.11 1.00 1.09 1.00 1.06 0.98 1.05 1.00 Normal 353 1.05 1.05 0.97 1.06 0.92 1.04 0.94 0.99 1.00 Normal 354 0.98 1.09 1.03 1.06 1.02 1.01 1.04 1.06 1.00 Normal 355 0.94 1.06 0.99 1.04 1.00 1.07 1.02 1.02 1.Q0 Normal 356 0.96 0.99 1.01 0.99 1.00 1.01 1.02 1.08 1.00 Normal 357 0.98 1.01 1.03 1.01 1.00 0.96 1.05 1.03 1.00 Normal 358 1.14 1.11 1.01 1.06 0.98 1.10 0.96 1.06 1.00 Normal 359 1.04 1.00 1.01 1.09 0.98 1.04 0.96 1.04 1.00 Normal 360 1.02 0.97 0.99 1.02 0.99 1.01 0.93 0.93 1.00 Normal 361 1.02 1.01 1.06 0.97 0.97 1.00 1.02 0.95 1.Q0 Normal 362 1.05 1.01 1.01 0.95 0.98 0.94 0.98 0.93 1.Q0 Normal 363 1.02 1.01 1.06 1.02 1.02 1.01 1.00 1.03 1.00 Normal

364 1.00 1.01 1.08 1.04 0.92 0.90 1.07 1.06 1.00 Normal 365 0.98 1.00 1.06 0.97 1.02 0.97 1.05 0.94 1.00 Normal 366 1.05 1.09 1.06 1.01 1.08 0.95 1.09 1.04 1.00 Normal 367 1.01 1.10 1.06 0.98 1.02 1.53 1.00 1.00 1.00 aneuploid (+C6) 368 1.00 0.97 1.02 0.95 0.98 0.99 1.04 0.97 1.00 Normal 369 1.00 1.00 1.07 1.05 1.01 0.99 1.02 1.03 1.Q0 Normal 370 0.99 0.98 0.95 0.98 0.97 1.03 0.94 0.98 1.00 Normal 371 0.96 0.95 0.94 0.96 0.99 1.08 0.90 0.91 1.00 Normal 372 1.02 1.04 1.06 0.99 1.04 1.00 0.88 0.99 1.00 Normal 373 0.96 1.01 0.99 0.99 0.99 0.91 1.07 0.99 1.00 Normal 374 0.98 0.99 0.97 0.96 0.96 1.04 0.96 0.94 1.00 Normal 375 1.02 1.06 1.10 1.02 1.08 0.97 1.10 1.03 1.00 Normal 376 1.02 1.04 1.01 1.09 1.05 0.99 1.07 1.02 1.00 Normal 377 1.30 0.99 1.01 1.00 0.99 1.06 1.01 0.94 1.00 aneuploid (+C1) 378 0.98 0.97 0.95 1.01 1.00 1.13 0.95 0.99 1.00 Normal 379 0.98 0.95 D.91 0.97 0.96 1.02 0.89 0.92 1.00 Normal 380 1.00 0.99 0.91 0.99 0.90 0.95 0.89 0.97 1.00 Normal 381 0.99 0.97 0.97 0.95 0.96 0.92 1.00 0.96 1.00 Normal 382 0.95 0.91 0.93 0.91 0.94 0.97 0.94 0.91 1.00 Normal 383 1.02 1.00 0.98 0.97 0.94 0.93 0.98 0.96 1.00 Normal 384 1.00 1.01 1.01 1.00 1.05 0.93 1.06 1.00 1.00 Normal 385 1.01 0.98 1.00 0.96 1.04 1.04 0.99 0.94 1.00 Normal 386 1.04 1.01 1.01 0.99 1.02 1.04 1.01 0.98 1.00 Normal

387 0.98 0.97 0.95 0.95 0.99 1.07 0.95 0.91 1.00 Normal 388 0.99 0.93 0.98 0.98 0.86 0.95 1.02 0.99 1.00 Normal 389 0.97 1.04 1.00 1.04 1.00 1.09 0.98 0.99 1.00 Normal 390 1.03 0.97 0.99 1.01 1.00 1.09 0.94 0.99 1.00 Normal 391 1.03 1.60 1.03 1.03 1.07 1.05 1.04 1.06 1.00 aneuploid (+C2) 392 0.96 0.95 0.98 0.95 0.98 0.91 0.90 0.93 1.00 Normal 393 1.01 1.02 1.09 1.05 1.02 0.91 1.07 1.11 1.00 Normal 394 1.02 0.99 0.97 0.97 1.02 0.93 0.98 0.95 1.00 Normal 395 1.02 1.06 0.99 1.05 1.03 1.02 0.98 1.01 1.00 Normal 396 0.95 1.03 0.98 0.99 1.00 0.93 1.05 1.11 1.00 Normal 397 1.02 1.04 1.01 1.04 1.01 1.13 1.05 1.02 1.00 Normal 398 0.94 0.93 0.91 0.91 0.99 1.08 0.90 0.89 1.00 Normal 399 1.05 0.97 1.01 1.03 1.03 1.12 1.00 0.97 1.00 Normal

[Table 3-7]

SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR SYBR C1 C2 C3 C4 C5 C6 C7 C8 C9 С1/С9 С2/С9 С3/С9 С4/С9 С5/С9 С6/С9 С7/С9 С8/С9 С9/С9 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 400 1.02 0.95 0.99 1.06 0.98 1.02 1.00 0.99 1.00 Normal 401 1.06 0.98 0.99 1.02 1.06 1.04 0.97 0.99 1.00 Normal 402 0.96 1.03 1.08 0.97 1.02 0.96 1.03 0.99 1.00 Normal 403 1.02 1.01 1.03 1.00 1.02 1.06 1.00 0.94 1.00 Normal

404 1.07 1.06 1.04 1.03 1.01 1.01 1.02 1.01 1.00 Normal 405 1.04 0.94 0.96 0.92 0.96 0.94 0.98 0.99 1.00 Normal 406 1.10 1.09 1.01 1.04 1.05 1.10 1.02 1.05 1.00 Normal 407 1.00 0.99 0.99 0.95 1.04 1.04 1.02 0.97 1.00 Normal 408 1.01 0.98 0.97 1.06 1.02 1.05 1.02 1.09 1.00 Normal 409 1.03 0.99 0.99 1.03 0.94 1.06 0.96 0.97 1.00 Normal 410 1.01 0.97 0.99 0.97 1.00 1.01 0.99 0.96 1.00 Normal 411 1.00 1.02 1.02 1.00 1.02 1.04 0.97 0.96 1.00 Normal 412 1.04 0.99 1.06 1.07 1.07 1.09 1.05 1.12 1.00 Normal 413 1.03 1.63 1.02 1.01 1.03 1.05 1.06 1.01 1.00 aneuploid(+C2) 414 1.01 0.98 0.96 1.00 0.98 1.07 0.93 1.00 1.00 Normal 415 1.04 1.06 1.05 1.03 1.05 0.95 1.01 1.00 1.00 Normal 416 0.99 1.42 0.90 1.01 0.95 0.98 0.90 0.97 1.00 aneuploid (+C2) 417 1.10 1.03 0.95 1.13 0.99 1.15 0.92 0.98 1.00 Normal 418 1.00 1.03 0.99 1.06 0.97 1.08 0.92 1.00 1.00 Normal 419 0.96 0.95 1.00 0.98 0.98 1.02 0.96 0.91 1.00 Normal 420 1.06 0.93 0.95 1.00 0.96 1.05 0.94 0.87 1.00 Normal 421 0.99 0.93 0.97 1.06 0.95 1.07 0.92 1.00 1.00 Normal 422 0.97 0.97 1.00 1.14 1.02 1.07 0.94 0.99 1.00 Normal

423 0.62 0.63 0.65 0.66 0.65 0.69 0.66 0.66 1.00 aneuploid (+C9) 424 0.99 0.97 1.01 1.01 1.03 1.05 0.98 0.91 1.00 Normal 425 1.00 1.05 1.01 1.07 1.03 1.12 0.96 1.05 1.00 Normal 426 1.02 0.97 0.97 1.00 1.02 1.04 0.95 0.96 1.00 Normal 427 0.97 1.0J 1.06 0.97 1.02 0.87 1.10 1.01 1.00 Normal 428 1.03 1.00 0.06 0.98 0.94 0.99 0.97 0.97 1.00 Normal 429 1.02 0.99 0.99 0.97 1.02 1.09 0.99 0.99 1.00 Normal 430 1.00 0.97 1.02 1.02 1.01 0.98 1.02 1.01 1.00 Normal 431 0.97 0.98 0.98 0.93 0.95 0.94 0.96 0.94 1.00 Normal 432 1.01 0.44 1.00 0.93 0.96 0.90 0.87 1.01 1.00 aneuploid (others) 433 1.01 1.03 0.96 0.99 0.96 0.93 0.96 1.02 1.00 Normal 434 0.98 0.95 0.99 0.91 1.01 0.93 1.05 0.97 1.00 Normal 435 1.02 1.01 0.93 1.01 0.97 1.02 0.96 1.03 1.00 Normal 436 0.97 0.98 1.01 0.91 0.99 1.01 1.01 1.10 1.00 Normal 437 0.96 0.97 0.99 0.95 1.00 0.99 1.00 0.95 1.00 Normal 438 0.98 0.97 0.97 0.93 0.97 0.98 0.98 0.99 1.00 Normal 439 1.09 .01 0.98 1.01 0.99 0.09 1.58 1.03 1.00 aneuploid (+C7) 440 0.95 0.94 1.05 0.98 0.96 0.97 1.01 1.00 1.00 Normal 441 0.93 0.89 0.95 0.88 0.99 0.94 0.96 0.98 1.00 Normal 442 0.99 1.06 0.99 1.06 1.00 1.08 0.98 1.02 1.00 Normal 443 1.02 1.08 1.04 1.06 1.01 1.07 1.00 1.06 1.00 Normal

444 0.93 0.95 1.00 0.93 1.02 0.93 1.07 0.99 1.00 Normal 445 1.00 0.95 1.03 0.91 1.01 0.88 1.02 1.01 1.00 Normal

[Table 4]

Table 4. Result of testing for aneuploid in F1 cultivar broccoli using the SYBR method (summary)

Number of plants Ratio (%) Normal 418 93.9 Aneuploid (+C1) 2 0.4 Aneuploid (+C2) five 1.1 Aneuploid (+C3) 0 0 Aneuploid (+C4) 1 0.2 Aneuploid (+C5) 0 0 Aneuploid (+C6) five 1.1 Aneuploid (+C7) five 1.1 Aneuploid (+C8) 0 0 Aneuploid (+C9) 3 0.7 Aneuploid (others) 6 1.3

Example 4 Atypical Cauliflower Plant Detection Example

Seeds of F1 cauliflower variety "SCF-30" developed by SAKATA SEED CORPORATION were sown in nursery trays. DNA from 654 individuals was extracted from newly emerged cotyledons from germinating seeds, followed by real-time PCR using a fluorescent probe method using markers specific to each chromosome.

Specifically, the above method was carried out as follows.

DNA was extracted from cauliflower cotyledons in the same manner as in Example 3 above.

The PCR test was performed using chromosome-specific markers divided into the following three combinations:

A triplex comprising a chromosome 6 marker, a chromosome 4 marker and a chromosome 2 marker;

A triplex comprising a chromosome 9 marker, a chromosome 3 marker and a chromosome 8 marker; and

A triplex containing a chromosome 1 marker, a chromosome 5 marker, and a chromosome 7 marker.

With regard to the chromosomal markers used in this document, the primers and probes shown in Tables 1 and 2 were used for each of the chromosomes. For example, a pair of primers having the sequences shown in SEQ ID NOs: 1 and 2 and a probe having the sequence shown in SEQ ID NO: 19 was used as a chromosome 1 marker.

As for real-time PCR, the same apparatus as in Example 3 was used, and PCR was performed under incubation conditions at 95°C for 1 minute, followed by 40 cycles of two-step PCR at 95°C for 15 seconds and 60°C. C for 45 seconds. Filters with an excitation wavelength of 465 nm, 533 nm, 618 nm and a detection wavelength of 510 nm, 580 nm and 660 nm were used to measure the FAM, HEX and Cy5 signals, respectively.

Based on the Ct value obtained by the second derivative method, the relative amount was calculated by the ΔΔCt method using the respective chromosome 2, chromosome 8 and chromosome 7 markers as endogenous controls.

The results were as shown in Table 5.

These results are summarized in Table 5 and thus it is clear that aneuploids are included in the ratio as shown in Table 6.

In addition, plants suspected to be aneuploids in this experiment were grown in the field to study the subsequent phenotype. Consistent with the case for broccoli, each of the aneuploids showed a characteristic appearance (Fig. 5) (the phenotypic characteristics of chromosomal trisomies were as shown in FIG. 7).

The above results show that, like broccoli, aneuploids appear in cauliflower, and this method is an efficient procedure for detecting these aneuploids in cauliflower.

Table 5. Aneuploid test result in F1 cauliflower varieties (raw data obtained by multiplex PCR based on the fluorescent probe method (value calculated using the ΔΔCt method))

[Table 5-1]

Triplex PCR С6С4С2 Triplex PCR С9С3С8 Triplex PCR С1С5С7 C6 C4 C2 C9 C3 C8 C1 C5 C7 С6/С2 С4/С2 С2/С2 С9/С8 С3/С8 C8/C8 С1/С7 С5/С7 С7/С7 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 1 0.98 0.99 1.00 1.00 1.02 1.00 1.04 0.98 1.00 Normal 2 0.99 1.02 1.00 0.94 1.05 1.00 0.96 1.09 1.00 Normal 3 0.75 0.73 1.00 1.11 1.07 1.00 0.94 0.98 1.00 Aneuploid (+C2) four 0.98 0.94 1.00 1.02 1.06 1.00 1.12 1.09 1.00 Normal five 1.03 1.02 1.00 0.99 0.89 1.00 0.78 0.77 1.00 Aneuploid (+C7) 6 0.95 0.97 1.00 0.98 1.03 1.00 1.06 1.11 1.00 Normal 7 0.97 1.02 1.00 1.02 0.88 1.00 1.06 1.05 1.00 Normal 8 0.98 1.01 1.00 1.03 0.96 1.00 0.98 1.00 1.00 Normal nine 0.96 0.96 1.00 1.02 0.97 1.00 0.98 1.00 1.00 Normal 10 0.90 0.96 1.00 1.00 0.93 1.00 1.08 1.13 1.00 Normal eleven 1.02 1.03 1.00 0.93 0.93 1.00 0.95 0.94 1.00 Normal 12 0.93 0.94 1.00 1.04 0.96 1.00 0.97 0.93 .00 Normal 13 0.93 1.04 1.00 1.09 1.00 1.00 0.91 0.95 1.00 Normal fourteen 0.98 1.06 1.00 1.03 1.00 1.00 0.98 0.96 1.00 Normal

15 1.01 1.02 1.00 1.03 1.02 1.00 1.09 1.02 1.00 Normal sixteen 0.95 1.00 1.00 1.02 1.01 1.00 0.96 1.00 1.00 Normal 17 1.09 1.05 1.00 0.98 0.87 1.00 1.03 0.98 1.00 Normal eighteen 0.99 0.93 1.00 1.01 0.95 1.00 0.96 0.99 1.00 Normal nineteen 1.05 1.06 1.00 1.05 1.01 1.00 1.03 1.06 1.00 Normal 20 1.06 1.09 1.00 1.05 1.02 1.00 J.03 0.96 1.00 Normal 21 0.94 0.98 1.00 0.88 1.03 1.00 J.13 1.06 1.00 Normal 22 0.90 0.91 1.00 0.98 1.01 1.00 0.98 1.03 1.00 Normal 23 1.06 1.06 1.00 1.03 1.03 1.00 1.06 1.03 1.00 Normal 24 1.11 1.05 1.00 1.01 1.08 1.00 1.01 1.01 1.00 Normal 25 0.97 0.94 4.00 0.98 1.01 1.00 0.98 0.98 1.00 Normal 26 1.02 1.00 1.00 0.89 0.93 1.00 1.24 0.95 1.00 Aneuploid (+C1) 27 0.95 0.95 1.00 1.00 0.93 1.00 1.01 0.98 1.00 Normal 28 0.97 1.00 1.00 1.09 0.97 1.00 1.03 1.02 1.00 Normal 29 0.96 0.96 1.00 1.00 0.97 1.00 1.06 0.97 1.00 Normal

thirty 0.96 1.00 1.00 1.07 0.99 1.00 1.01 0.94 1.00 Normal 31 1.12 1.08 1.00 0.96 0.93 1.00 1.04 1.00 1.00 Normal 32 0.96 0.99 1.00 0.98 0.93 1.00 1.04 0.94 1.00 Normal 33 1.01 1.00 1.00 1.02 0.96 1.00 0.96 0.98 1.00 Normal 34 0.94 1.06 1.00 0.99 0.86 1.00 1.12 0.98 1.00 Normal 35 1.01 1.04 1.00 0.98 0.99 1.00 1.03 0.95 1.00 Normal 36 0.93 0.95 1.00 1.00 0.99 1.00 1.01 1.05 1.00 Normal 37 0.99 0.98 1.00 1.04 1.01 1.00 1.01 0.99 1.00 Normal 38 0.96 0.98 1.00 1.00 1.00 1.00 1.10 0.94 1.00 Normal 39 0.97 1.01 1.00 1.04 1.09 1.00 0.88 0.95 1.00 Normal 40 1.03 1.05 1.00 0.98 0.95 1.00 0.92 0.98 1.00 Normal 41 1.05 0.98 1.00 0.96 0.94 1.00 1.03 0.98 1.00 Normal 42 1.03 1.06 1.00 0.96 1.01 1.00 0.92 0.98 1.00 Normal 43 1.03 0.99 1.00 1.04 0.95 1.00 0.98 0.92 1.00 Normal 44 1.08 0.95 1.00 1.00 0.97 1.00 0.97 0.93 1.00 Normal 45 1.02 0.98 1.00 1.00 0.99 1.00 1.03 1.04 1.00 Normal 46 0.93 0.94 1.00 1.00 0.96 1.00 0.89 1.03 1.00 Normal 47 1.01 1.05 1.00 0.98 0.89 1.00 0.92 0.96 1.00 Normal

48 0.92 0.90 1.00 0.94 0.91 1.00 1.01 1.03 1.00 Normal 49 0.98 0.91 1.00 0.98 0.99 1.00 1.13 1.03 1.00 Normal 50 0.93 0.96 1.00 0.96 0.99 1.00 1.01 1.02 1.00 Normal 51 1.00 1.00 1.00 0.95 0.99 1.00 0.95 0.96 1.00 Normal 52 1.02 1.08 1.00 0.96 0.93 1.00 0.96 0.99 1.00 Normal 53 1.01 1.04 1.00 0.94 1.04 1.00 1.01 1.06 1.00 Normal 54 1.01 1.03 1.00 1.02 1.07 1.00 0.96 0.99 1.00 Normal 55 1.12 1.11 1.00 0.98 1.00 1.00 1.02 1.05 1.00 Normal 56 1.03 0.98 1.00 0.99 0.97 1.00 0.93 0.94 1.00 Normal 57 0.71 0.71 1.00 1.05 1.09 1.00 0.97 1.01 1.00 Aneuploid (+C2) 58 0.97 1.00 1.00 0.98 1.01 1.00 0.94 0.94 1.00 Normal 59 0.98 0.97 1.00 1.02 1.11 1.00 1.02 1.04 1.00 Normal 60 1.03 1.01 1.00 1.04 1.00 1.00 1.01 1.05 1.00 Normal 61 0.99 1.02 1.00 0.95 0.97 1.00 1.08 1.12 1.00 Normal 62 0.94 0.97 1.00 1.05 1.09 1.00 1.01 1.02 1.00 Normal 63 0.95 1.02 1.00 0.94 0.99 1.00 1.07 1.05 1.00 Normal 64 0.96 1.00 1.00 0.98 1.01 1.00 1.01 1.02 1.00 Normal

65 1,Q 1.01 1.00 1.02 1.00 1.00 0.96 0.94 1.00 Normal 66 1.03 0.96 1.00 1.00 1.03 1.00 0.98 0.95 1.00 Normal 67 0.99 1.02 1.00 1.04 1.07 1.00 1.00 0.93 1.00 Normal 68 1.10 1.05 1.00 0.97 0.99 1.00 1.08 0.92 1.00 Normal 69 1.09 1.08 1.00 1.10 1.11 1.00 0.98 1.00 1.00 Normal

[Table 5-2]

Triplex PCR С6С4С2 Triplex PCR С9С3С8 Triplex PCR С1С5С7 C6 C4 C2 C9 C3 C8 C1 C5 C7 С6/С2 С4/С2 С2/С2 С9/С8 С3/С8 C8/C8 С1/С7 С5/С7 С7/С7 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 70 0.98 1.02 1.00 1.13 1.12 1.00 0.98 1.01 1.00 Normal 71 1.01 0.99 1.00 0.99 1.1z 1.00 1.04 1.06 1.00 Normal 72 0.93 0.98 1.00 1.02 1.09 1.00 0.96 0.92 1.00 Normal 73 1.03 1.00 1.00 1.05 1.09 1.00 1.03 1.07 1.00 Normal 74 1.05 1.09 1.00 0.94 1.01 1.00 1.01 1.02 1.00 Normal 70 0.89 0.98 1.00 1.07 1.04 1.00 0.93 0.g4 1.00 Normal

76 0.91 0.98 1.00 1.02 0.99 1.00 0.90 0.98 1.00 Normal 77 1.01 0.94 1.00 1.05 1.09 1.00 1.01 0.98 1.00 Normal 78 1.01 1.06 1.00 0.93 0.99 1.00 0.96 1.01 1.00 Normal 79 0.94 0.96 1.00 1.06 1.07 1.00 0.89 0.99 1.00 Normal 80 1.07 1.06 1.00 0.99 1.06 1.00 0.97 0.98 1.00 Normal 81 0.73 0.76 1.00 1.02 1.00 1.00 1.03 1.03 1.00 Aneuploid (+C2) 82 1.03 1.02 1.00 0.96 101 1.00 0.99 0.99 1.00 Normal 83 1.12 1.07 1.00 0.97 1.12 1.00 1.04 1.07 1.00 Normal 84 0.97 0.94 1.00 1.03 0.97 1.00 1.02 1.06 1.00 Normal 85 0.88 0.90 1.00 1.04 1.04 1.00 1.06 1.03 1.d0 Normal 86 1.06 1.06 1.00 0.97 0.91 1.00 1.04 1.10 1.00 Normal 87 0.94 0.96 1.00 1.00 1.00 1.00 0.97 1.01 1.00 Normal 88 1.37 1.04 1.00 0.93 0.89 1.00 0.89 0.98 1.00 Aneuploid (+C6) 89 1.04 1.00 1.00 1.03 1.01 1.00 0.97 0.95 1.00 Normal 90 1.06 1.02 1.00 0.98 0.95 1.00 0.91 0.99 1.00 Normal 91 1.11 1.05 1.00 0.94 0.95 1.00 1.02 1.06 1.00 Normal

92 0.94 0.95 1.00 1.06 0.99 1.00 0.97 0.90 1.00 Normal 93 0.91 0.98 1.00 1.04 0.93 1.00 1.05 0.96 1.00 Normal 94 1.05 1.12 1.00 1.04 1.02 1.00 1.01 1.02 1.00 Normal 95 0.92 0.94 1.00 1.00 1.03 1.00 0.93 1.01 1.00 Normal 96 1.00 1.04 1.00 0.96 1.03 1.00 0.91 0.99 1.00 Normal 97 0.98 1.02 1.00 1.00 1.01 1.00 0.93 0.99 1.00 Normal 98 1.01 1.00 1.00 1.09 0.93 1.00 0.93 0.90 1.00 Normal 99 1.09 1.10 1.00 1.12 1.09 1.00 1.01 0.96 1.00 Normal one hundred 1.01 1.00 1.00 0.97 0.95 1.00 0.98 0.97 1.00 Normal 101 1.06 1.05 1.00 1.01 0.99 1.00 1.01 1.02 1.00 Normal 102 0.98 0.96 1.00 1.05 1.00 1.00 0.88 0.96 1.00 Normal 103 0.97 0.99 1.00 0.97 0.97 1.00 1.05 1.11 1.00 Normal 104 0.98 1.00 1.00 1.01 1.06 1.00 1.09 1.02 1.00 Normal 105 0.94 1.01 1.00 0.96 1.03 1.00 0.93 0.98 1.00 Normal 106 1.02 1.05 1.00 1.01 1.09 1.00 1.03 1.04 1.00 Normal

107 0.95 0.94 1.00 1.28 1.10 1.00 1.03 1.08 1.00 aneuploid (+C9) 108 0.98 1.00 1.00 1.00 0.96 1.00 0.98 1.10 1.00 Normal 109 0.94 1.03 1.00 1.01 1.07 1.00 1.00 1.08 1.00 Normal 110 1.00 0.98 1.00 0.90 1.03 1.00 0.96 0.96 1.00 Normal 111 0.88 0.91 1.00 0.96 1.01 1.00 0.89 0.93 1.00 Normal 112 1.05 1.02 1.00 0.99 0.97 1.00 0.97 1.01 1.00 Normal 113 0.89 0.90 1.00 1.07 1.01 1.00 0.94 1.02 1.00 Normal 114 0.95 0.94 1.00 0.97 1.06 1.00 1.07 1.02 1.00 Normal 115 1.03 1.04 1.00 0.92 0.93 1.00 1.05 0.97 1.00 Normal 116 0.96 1.00 1.00 0.95 1.02 1.00 0.98 0.99 1.00 Normal 117 1.1 2 1.10 1.00 0.90 1.01 1.00 0.97 1.05 1.00 Normal 118 0.96 1.00 1.00 1.04 1.01 1.00 0.92 1.02 1.00 Normal 119 0.88 0.89 1.00 1.07 1.05 1.00 1.03 1.00 1.00 Normal 120 0.95 0.94 1.00 0.95 0.95 1.00 1.Q0 1.06 1.00 Normal 121 0.91 1.05 1.00 0.97 1.07 1.00 1.Q3 1.04 1.00 Normal 122 1.11 1.02 1.00 1.03 0.96 1.00 0.92 0.96 1.00 Normal 123 1.03 0.98 1.00 1.00 1.11 1.00 0.98 1.07 1.00 Normal

124 1.02 0.98 1.00 1.11 1.09 1.00 1.Q0 1.05 1.00 Normal 125 1.12 1.06 1.00 1.05 1.01 1.00 0.36 0.94 1.00 Normal 126 1.09 1.11 1.00 0.94 0.95 1.00 1 13 1.03 1.00 Normal 127 0.93 1.00 1.00 1.01 0.95 1.00 1.09 1.12 1.00 Normal 128 1.32 1.00 1.00 0.98 1.01 1.00 1.03 0.91 1.00 aneuploid(+C6) 129 1.01 0.99 1.00 0.98 0.98 1.00 0.99 1.05 1.00 Normal 130 1.01 1.00 1.00 0.95 1.05 1.00 1.01 1.06 1.00 Normal 131 1.01 1.02 1.00 1.00 0.88 1.00 0.96 0.94 1.00 Normal 132 1.04 1.02 1.00 0.92 1.00 1.00 1.09 1.12 1.00 Normal 133 1.12 1.07 1.00 1.02 1.04 1.00 1.05 0.99 1.00 Normal 134 1.04 1.07 1.00 1.03 0.99 1.00 0.98 1.03 1.00 Normal 135 1.08 1.03 1.00 0.91 0.94 1.00 1.08 0.98 1.00 Normal 136 1.04 1.02 1.00 0.98 0.95 1.00 0.98 0.97 1.00 Normal 137 0.94 0.96 1.00 1.03 1.08 1.00 1.02 0.96 1.00 Normal 138 1.00 1.05 1.00 0.99 1.01 1.00 0.95 1.03 1.00 Normal 139 1.04 1.04 1.00 1.01 1.06 1.00 1.10 1.11 1.00 Normal 140 0.92 0.95 1.00 1.01 0.99 1.00 1.01 1.03 1.00 Normal

[Table 5-3]

Triplex PCR С6С4С2 Triplex PCR С9С3С8 Triplex PCR С1С5С7

C6 C4 C2 C9 C3 C8 C1 C5 C7 С6/С2 С4/С2 С2/С2 С9/С8 С3/С8 C8/C8 С1/С7 С5/С7 С7/С7 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 141 0.94 0.93 1.00 1.00 1.04 1.00 0.99 0.96 1.00 Normal 142 1.04 1.06 1.00 1.00 1.00 1.00 0.99 1.03 1.00 Normal 143 0.99 0.96 1.00 1.14 1.04 1.00 0.99 1.08 1.00 Normal 144 0.98 1.00 1.00 0.99 0.95 1.00 0.97 1.02 1.00 Normal 145 1.06 1.08 1.00 1.10 1.01 1.00 0.95 1.03 1.00 Normal 146 0.92 0.99 1.00 0.98 0.99 1.00 1.05 1.00 1.00 Normal 147 0.98 0.98 1.00 1.00 1.04 1.00 0.95 0.96 1.00 Normal 148 0.94 0.98 1.00 1.02 1.01 1.00 1.06 1.06 1.00 Normal 149 0.99 0.92 1.00 0.96 1.10 1.00 1.02 1.05 1.00 Normal 150 1.06 1.07 1.00 0.99 1.01 1.00 1.01 0.93 1.00 Normal 151 1.03 0.98 1.00 1.10 1.09 1.00 1.03 1.02 1.00 Normal 152 0.96 1.01 1.00 1.09 1.03 1.00 1.09 1.06 1.00 Normal 153 1.06 1.08 1.00 1.02 0.95 1.00 1.01 1.02 1.00 Normal 154 0.98 1.05 1.00 1.10 1.09 1.00 0.96 0.96 1.00 Normal

155 0.99 0.98 1.00 0.95 0.94 1.00 1.02 0.98 1.00 Normal 156 1.04 0.98 1.00 1.03 1.10 1.00 0.97 0.99 1.00 Normal 157 1.00 1.03 1.00 0.90 0.95 1.00 1.01 0.94 1.00 Normal 158 1.06 1.02 1.00 0.99 1.03 1.00 0.99 1.09 1.00 Normal 159 0.93 0.96 1.00 0.94 0.98 1.00 0.93 0.94 1.00 Normal 160 0.98 0.95 1.00 1.03 1.01 1.00 1.01 0.96 1.00 Normal 161 0.97 0.98 1.00 0.98 1.03 1.00 0.91 0.95 1.00 Normal 162 1.02 0.98 1.00 1.05 1.12 1.00 1.00 1.05 1.00 Normal 163 1.01 1.05 1.00 0.90 0.95 1.00 1.06 1.05 1.00 Normal 164 0.91 0.92 1.00 0.98 0.98 1.00 0.96 0.96 1.00 Normal 165 1.03 1.05 1.00 1.03 1.13 1.00 0.96 0.92 1.00 Normal 166 0.88 0.94 1.00 1.00 0.93 1.00 1.05 0.99 1.00 Normal 167 0.88 1.00 1.00 0.95 0.97 1.00 1.01 0.93 1.00 Normal 168 0.97 1.00 1.00 0.99 0.97 1.00 0.97 0.92 1.00 Normal 169 0.94 0.92 1.00 0.97 0.89 1.00 0.96 0.93 1.00 Normal 170 1.03 1.02 1.00 1.05 1.01 1.00 1.29 0.95 1.00 aneuploid (+C1}

171 0.98 1.05 1.00 1.05 1.01 1.00 1.09 1.02 1.00 Normal 172 0.94 0.98 1.00 0.98 0.96 1.00 0.97 1.00 1.00 Normal 173 1.06 1.24 1.00 1.00 1.02 1.00 1.09 1.02 1.00 aneuploid (+C4) 174 1.00 1.08 1.00 0.92 0.97 1.00 1.06 1.10 1.00 Normal 175 1.08 1.08 1.00 1.01 0.99 1.00 1.04 0.95 1.00 Normal 176 1.03 1.11 1.00 1.01 0.94 1.00 0.99 0.96 1.00 Normal 177 0.96 1.01 1.00 0.96 0.96 1.00 0.98 1.00 1.00 Normal 178 1.01 1.05 1.00 0.97 0.95 1.00 1.06 1.00 1.00 Normal 179 0.87 0.96 1.00 1.00 0.99 1.00 1.01 1.02 1.00 Normal 180 1.08 1.05 1.00 1.02 0.97 1.00 0.98 1.00 1.00 Normal 181 0.96 1.04 1.00 0.92 1.02 1.00 0.94 0.93 1.00 Normal 182 1.04 1.08 1.00 1.10 1.12 1.00 0.88 0.96 1.00 Normal 183 1.04 1.04 1.00 0.99 1.07 1.00 1.11 0.98 1.00 Normal 184 1.03 1.03 1.00 1.07 1.08 1.00 1.01 0.99 1.00 Normal

185 0.94 0.94 1.00 0.94 0.97 1.00 1.02 1.00 1.00 Normal 186 1.01 1.00 1.00 0.99 0.97 1.00 1.08 1.02 1.00 Normal 187 1.01 0.98 1.00 1.00 0.94 1.00 1.01 1.05 1.00 Normal 188 1.12 1.06 1.00 1.13 1.10 1.00 0.99 1.03 1.00 Normal 189 1.04 1.03 1.00 1.00 0.99 1.00 1.06 1.02 1.00 Normal 190 1.04 1.00 1.00 0.96 0.90 1.00 0.99 0.97 1.00 Normal 191 1.09 1.11 1.00 1.01 0.96 1.00 1.08 1.01 1.00 Normal 192 1.10 1.09 1.00 1.02 0.99 1.00 1.00 1.00 1.00 Normal 193 1.06 1.00 1.00 0.96 0 98 1.00 0.96 1.00 1.00 Normal 194 0.99 0.99 1.00 1.05 0.90 1.00 1.07 1.11 1.00 Normal 135 0.96 1.07 1.00 0.99 0.94 1.00 1.02 1.03 1.00 Normal 196 1.09 1.06 1.00 1.00 0.95 1.00 0.99 1.02 1.00 Normal 197 0.92 0.94 1.00 0.99 0.96 1.00 0.91 0.94 1.00 Normal 198 1.05 1.04 1.00 1.03 1.01 1.00 0.96 0.98 1.00 Normal 199 0.93 0.98 1.00 0.94 0.94 1.00 1.02 0.98 1.00 Normal 200 1.07 1.01 1.00 0.95 0.99 1.00 1.00 1.03 1.00 Normal

201 1.03 1.03 1.00 1.05 1.01 1.00 1.01 1.04 1.00 Normal 202 1.01 1.06 1.00 1.05 0.95 1.00 1.02 1.11 1.00 Normal 203 1.06 1.10 1.00 1.01 0.95 1.00 0.97 1.01 1.00 Normal 204 1.06 1.04 1.00 1.05 1.01 1.00 1.03 1.00 1.00 Normal 205 0.95 0.98 1.00 0.98 0.94 1.00 0.97 0.95 1.00 Normal 206 1.06 1.06 1.00 1.01 0.97 1.00 1.03 1.02 1.00 Normal 207 1.00 1.00 1.00 1.13 1.08 1.00 0.98 1.00 1.00 Normal 208 0.91 0.94 1.00 1.00 1.01 1.00 1.02 0.98 1.00 Normal 203 1.01 0.99 1.00 0.98 0.95 1.00 1.03 1.02 1.00 Normal 210 1.06 1.00 1.00 0.99 1.07 1.00 0.93 0.91 1.00 Normal 211 0.94 0.90 1.00 0.93 0.97 1.00 1.05 1.00 1.00 Normal

[Table 5-4]

Triplex PCR С6С4С2 Triplex PCR С9С3С8 Triplex PCR С1С5С7 C6 C4 C2 C9 C3 C8 C1 C5 C7 С6/С2 С4/С2 С2/С2 С9/С8 С3/С8 C8/C8 С1/С7 С5/С7 С7/С7 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ∆∆Ct ΔΔCt ΔΔt ΔΔCt results 212 1.06 1.05 1.00 1.00 0.98 1.00 1.04 1.08 1.00 Normal 213 1.04 1.03 1.00 1.02 0.90 1.00 0.97 0.96 1.00 Normal 214 0.94 0.96 1.00 0.95 0.98 1.00 0.96 0.94 1.00 Normal

215 1.01 1.02 1.00 0.96 0.98 1.00 1.02 0.98 1.00 Normal 216 1.09 1.04 1.00 1.00 0.99 1.00 1.05 1.01 1.00 Normal 217 1.03 0.91 1.00 1.00 1.12 1.00 1.05 1.02 1.00 Normal 218 1.06 1.06 1.00 0.92 0.98 1.00 1.06 1.05 1.00 Normal 219 0.99 1.00 1.00 0.96 0.99 1.00 0.96 1.01 1.00 Normal 220 0.92 0.93 1.00 0.98 0.99 1.00 1.03 0.99 1.00 Normal 221 1.06 1.11 1.00 1.05 1.12 1.00 1.00 1.07 1.00 Normal 222 1.10 1.10 1.00 1.00 1.01 1.00 0.96 0.98 1.00 Normal 223 0.99 1.02 1.00 1.03 1.00 1.00 0.93 1.01 1.00 Normal 224 0.96 1.01 1.00 1.01 1.00 1.00 0.99 1.08 1.00 Normal 225 1.01 1.01 1.00 1.08 1.03 1.00 0.96 0.98 1.00 Normal 226 0.96 1.02 1.00 0.97 0.99 1.00 1.12 1.08 1.00 Normal 227 1.12 0.98 1.00 1.02 0.98 1.00 0.95 1.00 1.00 Normal 228 1.41 1.08 1.00 0.94 0.93 1.00 0.91 0.92 1.00 aneuploid (+C6) 229 1.00 0.99 1.00 0.90 0.98 1.00 1.01 1.01 1.00 Normal

230 0.93 0.89 1.00 0.98 1.01 1.00 1.05 1.02 1.00 Normal 231 1.08 1.10 1.00 1.00 0.97 1.00 1.06 1.11 1.00 Normal 232 1.01 1.07 1.00 1.03 1.06 1.00 0.96 0.99 1.00 Normal 233 1.03 1.08 1.00 0.94 1.06 1.00 0.93 1.00 1.00 Normal 234 0.97 1.05 1.00 0.96 1.02 1.00 0.97 0.93 1.00 Normal 235 1.07 0.98 1.00 0.90 0.89 1.00 1.12 1.08 1.00 Normal 236 1.03 1.02 1.00 1.02 0.94 1.00 0.95 0.98 1.00 Normal 237 1.03 1.08 1.00 1.05 1.09 1.00 0.95 1.05 1.00 Normal 238 1.08 1.07 1.00 1.01 1.06 1.00 0.98 0.98 1.00 Normal 239 0.96 1.02 1.00 1.02 1.09 1.00 1.01 0.98 1.00 Normal 240 1.00 1.05 1.00 1.02 1.00 1.00 1.06 0.92 1.00 Normal 241 0.92 0.96 1.00 1.00 1.09 1.00 0.96 0.99 1.00 Normal 242 0.94 0.99 1.00 0.98 1.01 1.00 1.09 0.98 1.00 Normal 243 0.93 0.94 1.00 1.01 0.97 1.00 1.02 0.99 1.00 Normal 244 0.96 1.00 1.00 1.02 1.01 1.00 0.94 0.98 1.00 Normal 245 1.02 1.05 1.00 0.94 0.96 1.00 1.03 0.92 1.00 Normal 246 0.91 1.01 1.00 1.00 1.13 1.00 0.96 0.94 1.00 Normal

247 1.10 1.08 1.00 0.98 1.01 1.00 1.02 0.97 ).00 Normal 248 0.96 0.96 1.00 1.07 1.03 1.00 1.04 1.10 1.00 Normal 249 0.98 1.02 1.00 0.99 0.98 1.00 1.08 0.98 1.00 Normal 250 1.03 1.00 1.00 1.07 1.00 1.00 1.D9 1.11 1.00 Normal 251 1.04 1.09 1.00 0.95 0.91 1.00 1.03 0.97 1.00 Normal 252 0.93 0.92 1.00 1.01 0.95 1.00 0.91 0.90 1.00 Normal 253 0.95 0.94 1.00 1.02 1.02 1.00 1.03 0.99 1.00 Normal 254 1.14 1.09 1.00 0.92 0.05 1.00 1.05 0.97 1.00 Normal 255 0.89 0.89 1.00 1.05 1.13 1.00 1.03 1.00 1.00 Normal 256 1.03 1.08 1.00 0.98 0.97 1.00 1.00 0.99 1.00 Normal 257 1.05 1.00 1.00 0.99 0.97 1.00 1.07 1.00 1.00 Normal 258 0.93 1.06 1.00 1.06 1.02 1.00 0.96 0.95 1.00 Normal 259 1.04 1.08 1.00 0.98 0.96 1.00 1.06 1.05 1.00 Normal 260 0.98 1.02 1.00 1.05 1.06 1.00 1.03 1.02 1.00 Normal 261 1.00 1.01 1.00 1.01 1.0J 1.00 1.03 1.05 1.00 Normal 262 0.89 0.96 1.00 \.02 0.97 1.00 1.01 0.98 1.00 Normal 263 1.03 1.05 1.00 1.10 1.05 1.00 1.03 1.02 1.00 Normal

264 0.94 1.03 1.00 1.04 0.08 1.00 1.03 1.05 1.00 Normal 265 1.02 1.02 1.00 0.96 0.90 1.00 0.94 0.94 1.00 Normal 266 0.91 0.92 1.00 0.96 0.97 1.00 0.98 0.92 1.00 Normal 267 0.91 1.02 1.00 0.96 1.05 1.00 1.01 0.96 1.00 Normal 268 0.96 0.96 1.00 1.04 1.10 1.00 0.96 1.01 1.00 Normal 269 1.01 1.04 1.00 1.03 1.04 1.00 0.98 1.00 1.00 Normal 270 0.98 1.00 1.00 1.10 1.01 1.00 1.04 1.05 1.00 Normal 271 0.94 0.93 1.00 1.00 1.02 1.00 0.89 0.86 1.00 Normal 272 1.02 1.08 1.00 1.01 0.91 1.00 0.93 0.96 1.00 Normal 273 0.94 0.95 1.00 1.00 0.97 1.00 0.93 0.96 1.00 Normal 274 1.02 1.08 1.00 1.10 1.00 1.00 1.03 0.96 1.00 Normal 275 0.98 0.97 1.00 0.92 0.95 1.00 1.01 0.98 1.00 Normal 276 1.03 0.99 1.00 0.98 0.93 1.00 1.01 1.06 1.00 Normal 277 0.98 1.00 1.00 0.99 0.99 1.00 1.06 1.05 1.00 Normal 278 1.12 1.01 1.00 0.95 0.97 1.00 1.00 0.95 1.00 Normal 279 1.06 0.99 1.00 1.01 1.05 1.00 1.01 1.01 1.00 Normal

280 1.01 1.02 1.00 0.98 0.94 1.00 0.96 0.98 1.00 Normal 281 1.03 1.00 1.00 1.00 0.93 1.00 0.96 1.01 1.00 Normal 282 1.04 0.99 1.00 1.06 1.06 1.00 0.94 0.94 1.00 Normal

[Table 5-5]

Triplex PCR С6С4С2 Triplex PCR С9С3С8 Triplex PCR С1С5С7 C6 C4 C2 C9 C3 C8 C1 C5 C7 С6/С2 С4/С2 С2/С2 С9/С8 С3/С8 C8/C8 С1/С7 С5/С7 С7/С7 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 283 0.97 0.96 1.00 0.92 0.90 1.00 1.07 1.05 1.00 Normal 284 1.12 1.04 1.00 0.99 0.98 1.00 0.91 0.95 1.00 Normal 285 1.01 0.96 1.00 0.98 0.93 1.00 0.97 0.94 1.00 Normal 286 1.05 1.00 1.00 0.96 1.09 1.00 0.88 0.96 1.00 Normal 287 1.06 1.03 1.00 0.89 0.90 1.00 0.98 1.00 1.00 Normal 288 0.89 0.92 1.00 1.02 1.01 1.00 1.06 0.98 1.00 Normal 289 0.89 0.89 1.00 0.97 0.95 1.00 0.95 0.99 1.00 Normal 290 0.94 0.89 1.00 1.04 0.97 1.00 1.02 0.94 1.00 Normal

291 1.03 1.03 1.00 1.01 1.01 1.00 0.98 0.98 1.00 Normal 292 0.96 0.99 1.00 1.01 1.06 1.00 0.88 0.98 1.00 Normal 293 0.91 0.94 1.00 0.97 0.99 1.00 0.98 1.05 1.00 Normal 294 1.06 1.06 1.00 1.07 1.03 1.00 0.95 0.94 1.00 Normal 295 0.94 0.87 1.00 0.95 0.93 1.00 1.08 0.98 1.00 Normal 296 1.06 1.02 1.00 1.04 0.96 1.00 1.00 0.94 1.00 Normal 297 0.98 1.00 1.00 0.94 0.87 1.00 1.03 1.07 1.00 Normal 298 1.03 1.00 1.00 1.12 1.09 1.00 1.02 1.02 1.00 Normal 299 0.98 0.96 1.00 1.00 1.05 1.00 0.93 0.96 1.00 Normal 300 0.69 0.75 1.00 0.98 0.99 1.00 1.09 1.07 1.00 aneuploid(+C2) 301 0.98 0.97 1.00 0.87 0.94 1.00 1.01 1.11 1.00 Normal 302 1.01 1.02 1.00 1.01 0.99 1.00 0.98 0.97 1.00 Normal 303 1.01 1.05 1.00 0.96 1.08 1.00 0.99 1.02 1.00 Normal

304 1.03 1.03 1.00 1.02 0.99 1.00 1.03 1.02 1.00 Normal 305 1.00 0.98 1.00 0.99 1.03 1.00 0.94 0.93 1.00 Normal 306 0.93 0.99 1.00 0.98 1.03 1.00 0.99 1.01 1.00 Normal 307 0.94 0.08 1.00 1.04 1.05 1.00 0.94 1.00 1.00 Normal 308 1.03 1.03 1.00 1.00 0.98 1.00 1.01 0.98 1.00 Normal 309 1.01 1.06 1.00 1.03 1.00 1.00 1.01 0.97 1.00 Normal 310 1.31 1.07 1.00 0.98 1.01 1.00 1.04 1.07 1.00 aneuploid (+c6) 311 0.98 1.00 1.00 1.01 1.03 1.00 1.07 1.04 1.00 Normal 312 0.94 0.94 1.00 0.97 1.01 1.00 1.01 1.00 1.00 Normal 313 1.07 1.03 1.00 1.04 0.96 I.00 1.04 1.06 1.00 Normal 314 1.06 1.08 1.00 1.02 1.06 1.00 0.96 1.00 1.00 Normal 315 1.03 0.98 1.00 1.00 1.05 1.00 0.99 1.06 1.00 Normal 316 1.00 1.04 1.00 0.96 1.00 1.00 0.95 0.93 1.00 Normal 317 1.06 0.94 1.00 0.96 0.98 1.00 0.91 0.99 1.00 Normal 318 1.01 1.02 1.00 0.96 1.04 1.00 0.95 0.94 1.00 Normal

319 1.00 0.98 1.00 1.03 0.88 1.00 0.96 0.92 1.00 Normal 320 0.95 0.92 1.00 1.07 1.04 1.00 1.03 0.97 1.00 Normal 321 1.07 0.99 1.00 1.00 0.98 1.00 0.98 0.87 1.00 Normal 322 1.03 0.99 1.00 1.08 1.07 1.00 1.00 0.97 1.00 Normal 323 1.05 0.98 1.00 0.98 1.08 1.00 0.91 0.96 1.00 Normal 324 0.98 0.92 1.00 1.03 1.04 1.00 1.06 0.99 1.00 Normal 325 1.08 1.04 1.00 1.00 0.95 1.00 0.98 0.98 1.00 Normal 326 0.99 1.02 1.00 1.06 1.02 1.00 1.06 0.94 1.00 Normal 327 0.98 0.97 1.00 0.98 0.99 1.00 0.97 1.00 1.00 Normal 328 1.05 0.98 1.00 1.01 1.11 1.00 1.04 0.97 1.00 Normal 329 1.06 1.01 1.00 1.04 0.98 1.00 0.90 0.98 1.00 Normal 330 1.03 0.39 1.00 1.01 1.03 1.00 0.89 0.94 1.00 Normal 331 0.98 0.97 1.00 1.04 0.95 1.00 0.93 0.98 1.00 Normal 332 1.03 1.01 1.00 0.96 1.04 T.00 0.98 1.01 1.00 Normal 333 1.09 1.00 1.00 1.03 1.07 1.00 1.02 0.96 1.00 Normal 334 1.02 0.99 1.00 0.94 1.07 1.00 0.91 0.98 1.00 Normal

335 0.97 0.98 1.00 0.97 0.90 1.00 0.97 1.02 1.Q0 Normal 336 0.91 0.96 1.0U 0.99 1.02 1.00 0.99 1.00 1.00 Normal 337 1.02 0.98 1.00 1.10 1.05 1.00 1.09 0.99 1.00 Normal 338 0.92 0.93 1.00 1.07 0.89 1.00 0.99 1.04 1.00 Normal 339 0.99 1.03 1.00 0.99 0.97 1.00 1.09 0.96 1.00 Normal 340 1.00 1.07 1.00 1.02 0.99 1.00 0.97 1.00 1.00 Normal 341 1.01 0.98 1.00 1.05 1.01 1.00 0.93 0.86 1.00 Normal 342 0.96 1.04 1.00 0.98 0.90 1.00 0.94 0.95 1.00 Normal 343 1.11 1.08 1.00 0.07 1.02 1.00 0.95 1.00 1.00 Normal 344 0.97 1.01 1.00 0.98 0.93 1.00 1.04 0.99 1.00 Normal 345 0.88 1.03 1.00 1.01 0.90 1.00 1.04 1.03 1.00 Normal 346 0.95 1.07 1.00 0.98 0.90 1.00 1.04 0.97 1.00 Normal 347 0.93 0.92 1.00 1.01 0.95 1.00 1.09 1.10 1.00 Normal 148 0.87 0.91 1.00 1.05 1.00 1.00 0.96 0.98 1.00 Normal 349 0.95 1.01 1.00 1.02 1.00 1.00 0.96 0.98 1.00 Normal 350 0.94 0.91 1.00 0.93 0.99 1.00 1.06 1.08 1.00 Normal

351 0.98 1.03 1.00 0.97 0.06 1.00 0.97 1.02 1.00 Normal 352 0.90 0.96 1.00 1.02 0.97 1.00 0.98 0.96 1.00 Normal 353 1.31 1.07 1.00 1.00 0.92 1.00 1.09 1.08 1.00 aneuploid (+C6)

[Table 5-6]

Triplex PCR С6С4С2 Triplex PCR С9С3С8 Triplex PCR С1С5С7 C6 C4 C2 C9 C3 C8 C1 C5 C7 С6/С2 С4/С2 С2/С2 С9/С8 С3/С8 C8/C8 С1/С7 С5/С7 С7/С7 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 354 0.95 1.03 1.00 1.10 1.00 1.00 1.00 1.05 1.00 Normal 355 0.95 0.99 1.00 0.96 1.04 1.00 1.04 1.00 1.00 Normal 356 1.02 1.03 1.00 0.97 0.88 1.00 1.06 1.05 1.00 Normal 357 1.07 1.04 1.00 0.98 1.01 1.00 1.01 1.00 1.00 Normal 358 1.02 1.06 1.00 1.06 1.01 1.00 1.03 0.92 1.00 Normal 359 1.02 1.00 1.00 0.97 1.00 1.00 1.05 1.11 1.00 Normal 360 0.97 1.04 1.00 1.05 0.97 1.00 0.95 0.92 1.00 Normal 361 0.97 0.89 1.00 1.02 0.97 1.00 0.99 0.90 1.00 Normal 362 1.00 1.02 1.00 1.05 1.04 1.00 1.09 1.12 1.00 Normal 363 1.02 1.01 1.00 0.94 0.93 1.00 0.98 0.94 1.00 Normal 364 1.04 1.05 1.00 0.97 0.98 1.00 1.01 0.92 1.00 Normal 365 1.06 1.09 1.00 0.97 1.06 1.00 0.93 0.95 1.00 Normal

366 0.93 0.98 1.00 1.06 0.96 1.00 1.13 1.10 1.00 Normal 367 0.96 0.99 1.00 1.01 1.02 1.00 1.00 0.98 1.00 Normal 368 1.05 1.03 1.00 1.02 0.93 1.00 0.96 0.96 1.00 Normal 369 0.92 1.01 1.00 1.02 1.12 1.00 1.01 0.96 1.00 Normal 370 1.01 1.01 1.00 0.95 0.94 1.00 1.09 1.02 1.00 Normal 371 0.98 1.01 1.00 0.95 0.96 1.00 0.95 0.94 1.00 Normal 372 1.02 1.00 1.00 0.90 0.90 1.00 0.96 1.00 1.00 Normal 373 1.01 1.03 1.00 0.99 0.93 1.00 0.99 1.01 1.00 Normal 374 1.00 1.03 1.00 0.97 0.92 1.00 0.98 0.93 1.00 Normal 375 1.08 1.02 1.00 0.97 0.94 1.00 1.04 1.06 1.00 Normal 376 1.04 1.08 1.00 0.93 0.91 1.00 1.07 1.00 1.00 Normal 377 1.02 1.01 1.00 0.95 0.97 1.00 1.11 1.11 1.00 Normal 378 1.05 1.01 1.00 1.01 1.00 1.00 1.08 1.06 1.00 Normal

379 1.01 1.03 1.00 0.97 1.05 1.00 1.06 1.03 1.00 Normal 380 0.93 1.01 1.00 0.98 1.03 1.00 1.10 1.02 1.00 Normal 381 1.45 1.06 1.00 1.00 1.00 1.00 0.93 1.00 1.00 aneuploid (+C6) 382 0.95 0.96 1.00 0.99 0.90 1.00 0.97 1.08 1.00 Normal 383 1.06 1.01 1.00 0.99 1.01 1.00 0.99 0.96 1.00 Normal 384 1.07 1.01 1.00 1.00 1.06 1.00 1.00 1.00 1.00 Normal 385 0.94 0.90 1.00 0.98 1.04 1.00 1.03 1.04 1.00 Normal 386 0.97 0.96 1.00 0.93 0.91 1.00 1.01 0.99 1.00 Normal 387 0.93 1.02 1.00 1.00 1.01 1.00 0.91 0.94 1.00 Normal 388 1.02 0.97 1.00 1.00 0.95 1.00 1.12 1.00 1.00 Normal 389 1.04 1.03 1.00 1.02 0.96 1.00 0.93 0.94 1.00 Normal 390 1.04 1.01 1.00 1.03 1.07 1.00 1.04 1.07 1.00 Normal 391 1.02 1.08 1.00 1.05 1.02 1.00 1.00 0.96 1.00 Normal 392 0.98 0.91 1.00 1.02 0.92 1.00 1.05 0.95 1.00 Normal 393 0.91 0.89 1.00 0.98 1.07 1.00 1.00 0.94 1.00 Normal

394 0.99 1.02 1.00 1.02 1.01 1.00 1.27 1.05 1.00 aneuploid (+C1) 395 0.90 1.00 1.00 0.98 1.02 1.00 1.00 1.02 1.00 Normal 396 0.93 0.99 1.00 1.04 1.05 1.00 1.02 0.98 1.00 Normal 397 1.04 0.98 1.00 1.01 0.97 1.00 1.03 0.98 1.00 Normal 398 0.97 0.96 1.00 1.00 1.00 1.00 1.30 1.08 1.00 aneuploid (+C1) 399 0.97 0.97 1.00 1.06 1.10 1.00 1.03 1.08 1.00 Normal 400 0.96 1.00 1.00 1.06 0.97 1.00 1.00 0.98 1.00 Normal 401 0.97 1.01 1.00 1.01 1.00 1.00 1.04 1.03 1.00 Normal 402 0.99 0.96 1.00 0.90 0.97 1.00 0.95 0.98 1.00 Normal 403 1.00 1.03 1.00 1.05 0.96 1.00 1.09 1.13 1.00 Normal 404 1.14 1.08 1.00 0.99 0.96 1.00 0.95 0.98 .00 Normal 405 1.10 0.96 1.00 1.04 0.97 1.00 0.95 1.00 1.00 Normal 406 0.97 1.02 1.00 1.03 1.07 1.00 1.07 1.00 1.00 Normal 407 0.99 1.01 1.00 1.00 1.00 1.00 1.04 0.95 1.00 Normal 408 0.93 0.89 1.00 1.01 1.01 1.00 1.00 0.99 1.00 Normal 409 1.01 0.95 1.00 0.99 0.97 1.00 0.97 0.94 1.00 Normal

4J0 1.04 1.03 1.00 0.99 0.98 1.00 0.96 1.00 1.00 Normal 4J1 1.02 1.02 1.00 0.93 0.90 1.00 0.96 1.00 1.00 Normal 412 1.00 1.01 1.00 0.96 0.93 1.00 1.01 1.01 1.00 Normal 413 0.96 1.03 1.00 1.03 1.08 1.00 1.04 0.98 1.00 Normal 414 1.02 0.98 1.00 1.01 1.07 1.00 1.00 1.03 1.00 Normal 415 0.99 0.98 1.00 1.01 1.07 1.00 1.04 1.09 1.00 Normal 416 1.07 1.02 1.00 1.04 1.13 1.00 1.01 0.97 1.00 Normal 417 0.99 1.03 1.00 1.04 1.01 1.00 D.95 1.00 1.00 Normal 418 1.01 1.01 1.00 1.03 1.08 1.00 0.99 0.98 1.00 Normal 419 0.99 1.04 1.00 1.01 1.03 1.00 0.98 1.01 1.00 Normal 420 1.01 0.99 1.00 0.99 0.98 1.00 0.92 0.96 1.00 Normal 421 0.95 0.94 1.00 0.98 0.94 1.00 0.97 1.00 1.00 Normal 422 0.88 0.91 1.00 1.01 1.00 1.00 0.94 1.00 1.00 Normal 423 1.00 1.02 1.00 0.95 1.00 1.00 0.97 0.98 1.00 Normal 424 1.03 0.99 1.00 0.99 1.04 1.00 1.04 1.07 1.00 Normal

[Table 5-7]

Triplex PCR С6С4С2 Triplex PCR С9С3С8 Triplex PCR С1С5С7

C6 C4 C2 C9 C3 C8 C1 C5 C7 С6/С2 С4/С2 С2/С2 С9/С8 С3/С8 C8/C8 С1/С7 С5/С7 С7/С7 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 425 0.96 0.92 1.00 0.97 1.00 1.00 1.01 0.97 1.00 Normal 426 1.08 1.02 1.00 1.04 1.00 1.00 0.95 0.98 1.00 Normal 427 1.05 1.05 1.00 1.05 1.03 1.00 0.92 0.96 1.00 Normal 428 1.00 1.01 1.00 1.05 1.02 1.00 0.92 0.94 1.00 Normal 429 0.93 0.98 1.00 1.02 1.04 1.00 1.08 0.98 1.00 Normal 430 1.03 1.01 1.00 0.97 0.93 1.00 1.01 1.03 1.00 Normal 431 0.92 0.98 1.00 1.03 1.00 1.00 1.05 1.13 1.00 Normal 432 0.97 1.06 1.00 0.99 0.90 1.00 1.03 0.99 1.00 Normal 433 1.04 0.95 1.00 1.02 1.03 1.00 1.00 0.99 1.00 Normal 434 1.00 1.03 1.00 1.00 1.04 1.00 0.95 0.98 1.00 Normal 435 1.05 0.99 1.00 1.00 1.07 1.00 1.05 0.99 1.00 Normal 436 0.98 0.97 1.00 1.03 0.94 1.00 1.02 0.98 1.00 Normal 437 0.99 1.07 1.00 0.95 0.99 1.00 0.76 0.73 1.00 aneuploid (+C7) 438 1.07 1.01 1.00 1.03 1.02 1.00 0.95 1.00 1.00 Normal

439 1.06 1.01 1.00 1.02 0.88 1.00 1.00 1.05 1.00 Normal 440 0.93 0.96 1.00 0.97 0.97 1.00 0.68 0.93 1.00 aneuploid (others) 441 1.03 1.01 1.00 0.92 0.92 1.00 1.11 1.06 1.00 Normal 442 1.03 1.08 1.00 0.95 1.00 1.00 0.93 0.96 1.00 Normal 443 0.95 0.92 1.00 0.95 1.03 1.00 1.02 1.11 1.00 Normal 444 0.93 0.91 1.00 1.00 0.96 1.00 0.99 0.99 1.00 Normal 445 0.99 1.01 1.00 1.01 0.97 1.00 1.04 0.98 1.00 Normal 446 0.95 0.96 1.00 1.01 0.07 1.00 1.09 0.98 1.00 Normal 447 1.00 0.99 1.00 1.13 1.05 1.00 1.10 1.03 1.00 Normal 448 0.99 1.00 1.00 0.99 0.93 1.00 0.97 1.04 1.00 Normal 449 1.04 0.94 1.00 1.07 1.04 1.00 1.08 1.02 1.00 Normal 450 1.02 1.01 1.00 1.02 1.02 1.00 0.92 0.98 1.00 Normal 451 1.00 1.06 1.00 1.02 1.00 1.00 0.99 0.96 1.00 Normal 452 1.08 1.01 1.00 1.01 1.02 1.00 0.95 0.95 1.00 Normal 453 1.09 1.05 1.00 1.07 1.10 1.00 0.98 0.96 1.00 Normal 454 0.97 1.01 1.00 0.94 0.90 1.00 0.97 1.00 1.00 Normal 455 1.04 0.98 1.00 1.07 1.09 1.00 1.01 0.98 1.00 Normal 456 0.96 0.91 1.00 1.03 1.06 1.00 1.04 0.94 1.00 Normal

457 1.02 1.01 1.00 1.02 1.01 1.00 1.08 1.04 1.00 Normal 458 0.99 1.04 1.00 0.94 0.94 1.00 0.98 0.97 1.00 Normal 459 0.92 0.96 1.00 1.01 0.99 1.00 0.96 0.96 1.00 Normal 460 0.97 0.98 1.00 0.99 0.95 1.00 1.04 1.00 1.00 Normal 461 0.96 0.93 1.00 1.00 1.00 1.00 0.97 0.01 1.00 Normal 462 1.00 0.97 1.00 0.93 1.04 1.00 0.94 1.00 1.00 Normal 463 0.99 1.02 1.00 1.03 1.01 1.00 1.01 0.95 1.00 Normal 464 1.01 1.04 1.00 0.99 0.95 1.00 1.02 0.97 1.00 Normal 465 0.99 1.01 1.00 1.01 1.00 1.00 0.90 0.98 1.00 Normal 466 0.88 0.94 1.00 1.02 1.08 1.00 0.99 1.05 1.00 Normal 467 0.97 0.94 1.00 1.01 1.09 1.00 1.03 1.05 1.00 Normal 468 1.04 1.01 1.00 1.02 1.00 1.00 0.98 1.04 1.00 Normal 469 1.04 1.07 1.00 1.02 1.10 1.00 1.03 1.11 1.00 Normal 470 0.95 0.96 1.00 1.02 1.02 1.00 0.97 1.02 1.00 Normal 471 0.98 0.94 1.00 0.99 1.04 1.00 1.01 1.01 1.00 Normal 472 0.97 0.98 1.00 1.04 1.01 1.00 1.05 1.02 1.00 Normal 473 1.07 1.03 1.00 1.02 1.05 1.00 1.08 1.11 1.00 Normal 474 0.93 0.96 1.00 0.95 1.03 1.00 1.05 1.00 1.00 Normal 475 0.97 0.94 1.00 1.07 1.02 1.00 1.01 1.00 1.00 Normal 476 0.99 0.98 1.00 0.89 0.93 1.00 0.99 1.02 1.00 Normal

477 0.95 0.98 1.00 1.04 1.04 1.00 0.93 0.94 1.00 Normal 478 1.07 1.05 1.00 1.04 1.09 1.00 1.06 1.08 1.00 Normal 479 0.94 0.99 1.00 0.97 1.09 1.00 0.96 0.98 1.00 Normal 480 1.11 1.03 1.00 0.96 1.00 1.00 0.97 1.02 1.00 Normal 481 1.01 1.02 1.00 1.03 1.04 1.00 1.01 0.98 1.00 Normal 482 0.99 1.05 1.00 1.05 1.05 1.00 0.98 1.00 1.00 Normal 483 0.98 0.94 1.00 1.02 1.10 1.00 0.99 0.93 1.00 Normal 484 6.97 1.01 1.00 0.95 0.92 1.00 0.92 0.88 1.00 Normal 485 0.97 1.00 1.00 1.06 1.02 1.00 1.03 0.97 1.00 Normal 486 0.99 1.03 1.00 1.02 1.11 1.00 1.04 1.00 1.00 Normal 487 1.05 1.06 1.00 0.97 1.04 1.00 1.08 1.14 1.00 Normal 488 1.04 1.01 1.00 0.95 0.96 1.00 1.07 1.01 1.00 Normal 489 0.95 0.98 1.00 0.99 1.02 1.00 1.02 0.94 1.00 Normal 490 0.99 0.98 1.00 0.98 1.12 1.00 1.09 1.06 1.00 Normal 491 1.01 0.99 1.00 0.96 1.02 1.00 0.08 1.00 1.00 Normal 492 0.96 1.04 1.00 1.01 0.97 1.00 0.98 1.03 1.00 Normal 493 1.06 1.08 1.00 0.99 1.10 1.00 1.06 1.05 1.00 Normal 494 1.04 1.04 1.00 0.95 1.01 1.00 1.01 1.05 1.00 Normal 495 1.04 1.01 1.00 1.02 1.05 1.00 1.09 1.06 1.00 Normal

[Table 5-8]

Triplex PCR С9С3С8 Triplex PCR С9С3С8 Triplex PCR С1С5С7 C6 C4 C2 C9 C3 C8 C1 C5 C7 С6/С2 С4/С2 С2/С2 С9/С8 С3/С8 C8/C8 С1/С7 С5/С7 С7/С7 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 496 0.91 0.89 1.00 1.01 1.01 1.00 0.94 0.94 1.00 Normal 497 1.11 1.10 1.00 1.06 1.03 1.00 0.94 0.97 1.00 Normal 498 0.95 0.96 1.00 1.06 1.08 1.00 0.97 1.00 1.00 Normal 499 102 0 98 1.00 1.04 1.03 1.00 0.99 0.98 1.00 Normal 500 1.09 1.06 1.00 1.00 1.04 1.00 0.95 1.02 1.00 Normal 501 1.08 1.05 1.00 0.96 1.01 1.00 0.98 1.00 1.00 Normal 502 0.98 0.96 1.00 0.95 1.00 1.00 1.06 1.11 1.00 Normal 503 0.90 0.95 1.00 0.98 1.04 1.00 0.95 0.99 1.00 Normal 504 0.97 0.98 1.00 0.99 1.09 1.00 0.90 0.96 1.00 Normal 505 0.98 0.96 1.00 0.99 1.10 1.00 0.93 1.05 1.00 Normal 506 0.95 0.91 1.00 0.96 0.99 1.00 0.98 0.92 1.00 Normal 507 1.02 0.98 1.00 1.03 1.07 1.00 1.04 0.96 1.00 Normal 508 1.00 0.94 1.00 0.94 0.99 1.00 0.99 1.02 1.00 Normal 509 0.99 1.03 1.00 0.97 J.05 1.00 0.98 0.66 1.00 aneuploid (others) 510 0.68 0.71 1.00 1.02 1.04 1.00 0.98 1.00 1.00 aneuploid (+C2)

511 0.99 0.95 1.00 0.95 0.97 1.00 0.95 1.02 1.00 Normal 512 1.04 1.03 1.00 1.00 1.13 1.00 1.03 1.08 1.00 Normal 513 one hundred 1.04 1.00 0.95 0.98 1.00 0.96 1.00 1.00 Normal 514 0.96 0.96 1.00 0.95 0.94 1.00 0.92 0.96 1.00 Normal 515 108 1.03 1.00 1.04 1.03 1.00 0.95 1.05 1.00 Normal 516 1.02 1.08 1.00 0.90 1.05 1.00 1.00 1.01 1.00 Normal 517 1.05 0.96 1.00 1.01 0.90 1.00 0.97 0.96 1.00 Normal 518 0.94 0.97 1.00 0.95 0.92 1.00 0.90 0.93 1.00 Normal 519 1.02 1.07 1.00 1.01 0.92 1.00 0.97 0.95 1.00 Normal 520 1.04 1.03 1.00 1.14 1.07 1.00 0.99 1.05 1.00 Normal 521 1.01 1.03 1.00 1.00 0.95 1.00 0.99 1.05 1.00 Normal 522 1.09 1.01 1.00 0.99 0.92 1.00 0.95 0.98 1.00 Normal 523 0.99 1.01 1.00 1.04 1.05 1.00 1.03 0.98 1.00 Normal 524 1.07 1.09 1.00 1.02 0.95 1.00 1.04 1.02 1.00 Normal 525 1.07 1.08 1.00 1.03 0.96 1.D0 1.03 1.01 1.00 Normal 526 1.04 0.98 1.00 0.99 1.01 1.00 1.04 1.02 1.00 Normal 527 0.99 1.02 1.00 0.93 0.95 1.00 0.91 0.99 1.00 Normal 528 1.02 0.98 1.00 0.87 0.95 1.00 1.03 1.05 1.00 Normal 529 0.96 0.95 1.00 0.96 0.91 1.00 0.98 0.89 1.00 Normal 530 1.07 1.06 1.00 1.02 1.00 1.00 1.06 1.09 1.00 Normal

531 0.97 0.95 1.00 1.01 0.93 1.00 0.94 0.98 1.00 Normal 532 1.05 1.06 1.00 1.02 0.97 1.00 0.98 1.03 1.00 Normal 533 0.97 1.04 1.00 0.96 0.95 1.00 1.02 0.98 1.00 Normal 534 1.01 1.01 1.00 1.07 0.95 1.00 0.97 0.99 1.00 Normal 535 0.93 0.94 1.00 1.07 1.00 1.00 0.90 0.96 1.00 Normal 536 0.97 1.01 1.00 1.01 1.01 1.00 1.00 1.00 1.00 Normal 537 0 95 0.93 1.00 1.06 1.03 1.00 0.97 0.96 1.00 Normal 538 1.04 1.02 1.00 0.97 1.00 1.00 0.90 0.92 1.00 Normal 539 1.00 1.03 1.00 1.0T 0.89 1.00 1.04 1.09 1.00 Normal 540 0.99 1.01 1.00 0.95 0.98 1.00 0.91 1.00 1.00 Normal 541 1.03 1.01 1.00 0.99 0.99 1.00 1.04 1.04 1.00 Normal 542 1.02 0.97 1.00 1.03 1.01 1.00 1.00 1.03 1.00 Normal 543 1.08 1.04 1.00 0.97 1.00 1.00 0.93 0.95 1.00 Normal 544 0.91 0.92 1.00 0.97 0.95 1.00 1.01 1.03 1.00 Normal 545 1.02 0.96 1.00 1.02 1.14 1.00 0.93 0.94 1.00 Normal 546 1.02 0.98 1.00 0.95 0.94 1.00 1.04 1.05 1.00 Normal 547 0.94 1.01 1.00 0.93 0.98 1.00 0.99 1.05 1.00 Normal 548 0.99 1.01 1.00 0.94 1.02 1.00 0.94 0.99 1.00 Normal 549 0.95 0.98 1.00 0.97 0.99 .00 0.99 0.96 1.00 Normal 550 0.99 1.00 1.00 0.98 0.97 1.00 0.98 0.97 1.00 Normal 551 1.10 1.03 1.00 1.02 1.00 1.00 0.97 0.97 1.00 Normal 552 1.04 1.01 1.00 0.99 1.12 1.00 1.01 1.02 1.00 Normal 553 1.41 1.10 1.00 1.01 1.00 1.00 1.03 1.10 1.00 aneuploid (+C6)

554 1.04 1.03 1.00 0.93 1.01 1.00 0.95 0.99 1.00 Normal 555 1.02 1.00 1.00 1.04 1.05 1.00 0.98 0.99 1.00 Normal 556 1.03 1.01 1.00 0.99 1.04 1.00 0.99 1.00 1.00 Normal 557 0.99 1.02 1.00 1.02 1.07 1.00 0.95 0.94 1.00 Normal 558 0.93 0.96 1.00 1.02 1.0J 1.00 0.93 0.95 1.00 Normal 559 1.02 1.05 1.00 1.05 1.05 1.00 1.03 0.98 1.00 Normal 560 1.02 1.05 1.00 1.05 1.10 1.00 1.03 1.01 1.00 Normal 561 1.07 1.03 1.00 0.99 1.01 1.00 0.98 0.98 1.00 Normal 562 0.96 0.93 1.00 0.97 1.03 1.00 0.98 0.94 1.00 Normal 563 0 93 0.96 1.00 1.04 1.14 1.00 0.93 0.98 1.00 Normal 564 1.13 1.09 1.00 1.04 1.03 1.00 1.04 1.08 1.00 Normal

[Table 6]

Table 6. Result of testing for aneuploid in F1 cauliflower cultivar by TaqMan method (total)

Number of plants Ratio (%) Normal 542 96.1 Aneuploid (+C1) four 0.7 Aneuploid (+C2) five 0.9 Aneuploid (+C3) 0 0 Aneuploid (+C4) 1 0.2 Aneuploid (+C5) 0 0 Aneuploid (+C6) 7 1.2 Aneuploid (+C7) 2 0.4 Aneuploid (+C8) 0 0

Aneuploid (+C9) 1 0.2 Aneuploid (others) 2 0.4

Example 5 Atypical Plant Detection Example in Cabbage

Individuals with a morphology different from the normal appearance at the harvest stage were selected from the F1 cabbage cultivar "SCB-81" cultivated by SAKATA SEED CORPORATION. DNA is extracted from mature leaves of each plant and aneuploid analysis is carried out in the same manner as in Example 4 above.

The results were as shown in Table 7.

As a result, all trisomic plants were found except for chromosome 3, and it was found that chromosome aneuploidy also caused the majority of atypical individuals in cabbage.

These facts indicate that the method of the present invention is effective for the analysis of atypical individuals not only in broccoli and cauliflower, but also in cabbage.

FIG. 6 shows the appearance of various aneuploids (the phenotypic characteristics of chromosomal trisomies were as shown in FIG. 7). As shown in FIG. 6 and in Table 7, in addition to typical trisomies, there were also individuals in whom aneuploidy occurred on multiple chromosomes.

[Table 7]

Table 7. Aneuploid testing result in F1 cabbage cultivars (raw data obtained by multiplex PCR based on the fluorescent probe method (value calculated using the ΔΔCt method))

Triplex PCR С9С3С8 Triplex PCR С9С3С8 Triplex PCR С1С5С7 C6 C4 C2 C9 C3 C8 C1 C5 C7 С6/С2 С4/С2 С2/С2 С9/С8 С3/С8 C8/C8 С1/С7 С5/С7 С7/С7 individual no. ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt ΔΔCt results 1 1.03 1.03 1.00 0.95 0.95 1.00 1.04 1.03 1.00 Normal 2 0.96 0.98 1.00 0.99 1.03 1.00 1.02 0.95 1.00 Normal 3 1.04 1.03 1.00 0.97 1.02 1.00 1.00 1.05 1.00 Normal

four 0.96 0.96 1.00 1.04 0.94 1.00 0.94 0.97 1.00 Normal five 1.07 1.03 1.00 1.02 1.03 1.00 1.02 1.01 1.00 Normal 6 0.94 0.97 1.00 1.04 1.02 1.00 0.99 1.00 1.00 Normal 7 1.32 0.99 1.00 0.99 0.96 1.00 1.07 1.03 1.00 aneuploid (+C6) 8 1.05 1.01 1.00 1.04 0.95 1.00 0.75 0.76 1.00 aneuploid (+C7) nine 1.34 0.97 1.00 1.05 1.03 1.00 1.04 1.05 1.00 aneuploid (+C6) 10 1.02 1.38 1.00 1.01 1.03 1.00 1.05 0.99 1.00 aneuploid (+C4) eleven 0.99 0.98 1.00 1.03 1.05 1.00 1.32 1.07 1.00 aneuploid (+C1) 12 0.71 0.72 1.00 1.01 0.96 1.00 0.98 0.99 1.00 aneuploid (+C2) 13 0.74 0.75 1.00 0.95 1.04 1.00 1.30 1.03 1.00 aneuploid (+C1,+C2) fourteen 0.93 0.97 1.00 1.00 0.89 1.00 1.04 1.30 1.00 aneuploid (+c5) 15 0.72 0.72 1.00 1.00 0.95 1.00 1.00 0.99 1.00 aneuploid (+C2) sixteen 1.35 0.95 1.00 1.00 1.09 1.00 1.02 1.02 1.00 aneuploid (+C6) 17 0.96 0.98 1.00 0.74 0.79 1.00 1.03 1.02 1.00 aneuploid (+C8) eighteen 1.02 0.98 1.00 1.28 1.02 1.00 1.00 1.03 1.00 aneuploid (+C9) nineteen 0.75 0.74 1.00 1.00 0.96 1.00 1.04 1.03 1.00 aneuploid (+C2) 20 0.93 1.05 1.00 0.97 0.91 1.00 0.79 0.79 1.00 aneuploid (+C7) 21 0.97 1.00 1.00 1.01 1.07 1.00 1.30 1.05 1.00 aneuploid (+C1) 22 0.76 0.78 1.00 1.02 0.96 1.00 1.04 0.99 1.00 aneuploid (+C2) 23 1.31 1.05 1.00 0.97 0.92 1.00 1.07 1.06 1.00 aneuploid (+C6) 24 1.10 1.04 1.00 1.01 1.00 1.00 0.76 0.77 1.00 aneuploid (+C7) 25 1.02 0.97 1.00 0.76 0.80 1.00 1.24 0.99 1.00 aneuploid(+C1,+C8)

26 1.39 1.08 1.00 1.02 1.02 1.00 1.04 0.99 1.00 aneuploid (+C6) 27 0.98 0.96 1.00 1.02 1.06 1.00 1.31 1.06 1.00 aneuploid (+C1) 28 0.76 0.72 1.00 1.00 0.92 1.00 1.01 0.99 1.00 aneuploid (+C2) 29 0.98 1.01 1.00 0.78 0.81 1.00 1.04 1.07 1.00 aneuploid (+C8) thirty 0.77 0.73 1.00 1.04 0.98 1.00 1.01 0.99 1.00 aneuploid (+C2) 31 0.95 0.98 1.00 1.02 0.95 1.00 1.38 1.01 1.00 aneuploid (+C1) 32 0.93 1.04 1.00 0.96 0.88 1.00 1.07 1.31 1.00 aneuploid (+C5) 33 0.97 1.03 1.00 0.75 0.73 1.00 1.04 1.30 1.00 aneuploid(+C5,+C8) 34 0.99 0.92 1.00 0.73 0.82 1.00 1.28 1.04 1.00 aneuploid(+C1,+C8) 35 0.93 0.91 1.00 1.26 1.08 1.00 1.04 0.96 1.00 aneuploid (+C9) 36 1.04 1.35 1.00 1.03 0.95 1.00 1.02 1.01 1.00 aneuploid (+C4) 37 1.09 1.37 1.00 0.95 0.97 1.00 0.98 0.95 1.00 aneuploid (+C4)

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SEQUENCE LIST

<110> Sakata Seed Corporation

<120> METHOD FOR DETECTING AN ATYPICAL PLANT BRASSICA OLERACEA

<130> 800521JP01

<160> 27

<170>PatentIn version 3.5

<210> 1

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 1

ctggcaaatg taagcccttt ct 22

<210> 2

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 2

cttgtcttat tacagcagat gcattc 26

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 3

cgccattgct ttctctctac tct 23

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 4

gaagaggaag gactcgagga ag 22

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 5

cttaggattc gggttcgttt g 21

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 6

gccgtaagat ttcaaagaga cttc 24

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 7

cgtctcttgt ggtggttgaa g 21

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 8

tcaacttcat ctgcttggta atg 23

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 9

agcacatcat cccccatact t 21

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 10

cagtctctct ctccttgatg acg 23

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 11

aggaagagga aattgtcatt cg 22

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 12

gtgaccgttg cagcagataa 20

<210> 13

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 13

aagaaattag ccacaagtcg taaata 26

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 14

acgtgaatga tggatatttg atctc 25

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 15

aaagctcgtg aagcaaatac tacc 24

<210> 16

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 16

gaagcatacc aggagggaaa taa 23

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 17

gccatcgcga atcaaagata 20

<210> 18

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 18

atttggtatt ttgcaggcta cag 23

<210> 19

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> probe

<400> 19

cacttgtaaa acatgggttt gatcaaaaga 30

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> probe

<400> 20

tcctctactt ccaccccatc tgcc 24

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> probe

<400> 21

ccgatctgaa aagggagcta acgac 25

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> probe

<400> 22

ttgcagcaag gagcttagac cacag 25

<210> 23

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> probe

<400> 23

tctcgagaaa tctcatcgct gcttg 25

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> probe

<400> 24

ttctcagagc tgttccctcc tccac 25

<210> 25

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> probe

<400> 25

ttgcaccacc gttacctttt aacacaa 27

<210> 26

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> probe

<400> 26

tgttttgttt ggtgggcaaa tctctt 26

<210> 27

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> probe

<400> 27

tggagatctt ccacctcatc ttgga 25

<---

Claims (16)

1. A method for detecting an aneuploid plant Brassica oleracea, comprising: providing DNA markers that include one DNA marker that is specific to any one of chromosomes 1-9 of the Brassica oleracea plant and another DNA marker that is specific to the other chromosome from chromosomes 1-9 of the Brassica oleracea plant, performing real-time PCR using as a template DNA isolated from a sample obtained from a Brassica oleracea plant for testing, and DNA markers; and detection of chromosomal aneuploidy by the relative difference between the amplification values obtained using DNA markers. 2. The method of claim 1 further comprising determining whether the test plant is aneuploid on one of its chromosomes using DNA markers specific to each of chromosomes 1 to 9 of the Brassica oleracea plant. 3. The method according to claim 1 or 2, wherein the method uses as a DNA marker a primer that is specific for only one of the chromosomal DNA of the Brassica oleracea plant and can produce an amplification product in a PCR reaction when chromosomal DNA is present. 4. The method according to any one of paragraphs. 1-3, where the method uses as DNA markers (i) a primer that is specific for only one of the chromosomal DNA of the Brassica oleracea plant and can produce an amplification product in a PCR reaction when chromosomal DNA is present; and (ii) a probe that is specific for chromosomal DNA identical to any of the chromosomal DNAs described in (i) and can detect an amplification product obtained by a PCR reaction based on the primer described in (i). 5. The method according to claim 3 or 4, wherein the method uses an intercalator that binds to double-stranded DNA synthesized by the PCR reaction and emits fluorescence, or uses a probe modified with a fluorescent dye to emit fluorescence in the PCR elongation reaction. 6. The method according to any one of paragraphs. 1-5, where the method uses an increase in the fluorescent signal obtained by real-time PCR as an indicator for the detection of an aneuploid chromosome. 7. The method according to any one of paragraphs. 1-6, where the method uses one or more primers with the nucleotide sequences shown in SEQ ID NOs: 1-18 as a DNA marker. 8. The method according to any one of paragraphs. 1-7, wherein the method uses one or more primers with the nucleotide sequences shown in SEQ ID NOs: 1-18 and one or more probes with the nucleotide sequences shown in SEQ ID NOs: 19-27 as a DNA marker. 9. A set of primers for use in the method according to any one of paragraphs. 1-8, comprising at least one or more primers with the nucleotide sequences shown in SEQ ID NO: 1-18. 10. A set of primers and probes for use in the method according to any one of paragraphs. 1-8, including at least one or more primers with the nucleotide sequences shown in SEQ ID NO: 1-18; and at least one or more fluorescent dye-modified probes with the nucleotide sequences shown in SEQ ID NOs: 19-27. 11. A method for breeding a culture of Brassica oleracea, including an assessment of the frequency of occurrence of chromosome aneuploids for each genetic line of a Brassica oleracea plant using the method according to any one of paragraphs. 1-8 to select a line with a low incidence of aneuploids. 12. A method for quality control of seeds of Brassica oleracea, including testing to determine the degree of contamination with aneuploids contained in seeds from a batch of seeds of Brassica oleracea, using the method according to any one of paragraphs. 1-8. 13. A method for quality control of Brassica oleracea plants, including testing to determine the degree of contamination with aneuploids contained in Brassica oleracea plants, using the method according to any one of paragraphs. 1-8.

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