Rice transformation event GATV3-867-3 and its detection primers and applications

Abstract

The present invention relates to the field of bioengineering technology, and in particular to a rice transformation event GATV3-867-3, its detection primers, and applications. The right flank sequence of the flanking sequence is shown in SEQ ID NO.1; the left flank sequence of the flanking sequence is shown in SEQ ID NO.2. Specific primers for detecting the flanking sequences are shown in SEQ ID NO.9-10 and SEQ ID NO.11-12. PCR amplification is performed on a DNA sample of the rice to be tested using the specific primers, which can specifically indicate whether the T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted at bases 8873091-8873112 of the non-coding region of rice chromosome 5. The present invention successfully realizes the detection and safety management of transgenic rice and its derivative lines.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, in particular to a rice transformation event GATV3-867-3 and a detection primer and application thereof. Background Art

[0002] In recent years, genetically modified crops such as soybeans, corn, cotton, and rapeseed have been approved for cultivation and production in many countries. These crops can be processed into food, feed, or food additives. However, due to ongoing controversy surrounding the ecological and food safety of genetically modified products, strict regulation of these products is necessary. Biosafety assessment of genetically modified crops is a crucial component of genetically modified product regulation. This involves identifying the specific insertion site of the transformation event within the host genome, determining whether the insertion site is inactivating or deleting the host genome, and further estimating the impact of the transformation event on the host and potential safety issues.

[0003] Since the integration position of the exogenous insert in the host plant genome is random, the insertion site flanking sequences, which are formed by splicing the left and right end sequences of each exogenous insert with the host genome sequence, are unique. Therefore, the insertion site flanking sequences are unique identifiers that distinguish different transformation events and are important technical data for establishing specific detection methods for transgenic plant lines. The specific detection of transformation events is highly specific and can accurately identify different transgenic crop lines. Currently, the isolation of flanking sequences of exogenous inserts is mainly based on PCR technology. Established specific detection methods include inverse PCR, exogenous linker-mediated PCR, semi-random primer PCR, whole genome resequencing technology, etc. Among them, thermal asymmetric staggered PCR (Tail-PCR), high-efficiency thermal asymmetric PCR (hiTail-PCR) or chromosome walking (GenomeWalking) in semi-random primer PCR are currently commonly used methods.

[0004] Genetic Automation Technology (GAT) is a novel hybrid seed breeding technology that can successfully utilize recessive nuclear male sterile lines. The core concept of GAT is to utilize modern biotechnology to tightly link crop pollen fertility restoration genes, pollen abortion genes, herbicide sensitivity genes, and selection marker genes in a specific sequence and orientation on a GAT vector. These genes are then introduced into recessive nuclear male sterile lines through high-throughput gene transformation technology, resulting in a large number of transformation events. Summary of the Invention

[0005] Based on the genetically stable and agronomically excellent rice GAT transformation event GATV3-867-3, this study clarifies the molecular characteristics of the rice GAT transformation event GATV3-867-3 and advances its biosafety assessment. Using DNA from T0 generation GATV3-867-3 plants as a template, the study utilizes hiTail-PCR to isolate the T-DNA flanking sequences. Detection primers are designed based on the left and right end sequences and flanking sequences of the T-DNA. This method establishes a specific detection method for the rice GAT transformation event GATV3-867-3 and simultaneously tests its specificity and sensitivity, providing a technical basis for the detection and identification of the rice GAT transformation event GATV3-867-3 and its derivatives.

[0006] The rice GAT transformation event GATV3-867-3 was disclosed in a conference abstract (The 9th International Conference on Botany, Dec. 8-10, 2023, Characterization of GAT transformation events offered a promising option to GMS application in rice, Xiongxia Jin). The "867-3" mentioned in the abstract of the conference paper refers to this transformation event GATV3-867-3.

[0007] Based on this, the purpose of the present invention is to provide the flanking sequences of the exogenous vector insert fragment of the rice GAT transformation event GATV3-867-3, and to provide corresponding detection primers. Specifically:

[0008] First, the present invention provides the flanking sequence of the exogenous insertion vector of the transgenic rice transformation event GATV3-867-3, the right flank sequence of the flanking sequence is shown as SEQ ID NO.1; the left flank sequence of the flanking sequence is shown as SEQ ID NO.2.

[0009] In some embodiments, the flanking sequences are amplified using primer pairs as shown in SEQ ID NOs. 11-12 and 9-10, respectively.

[0010] Specifically, the right flank sequence is composed of bases 1 to 301 derived from chromosome 5 of the rice genome and bases 302 to 413 derived from the GATV3 vector sequence. The right flank sequence is the 3'-end border flanking sequence of the exogenous insertion vector of the rice GAT transformation event GATV3-867-3;

[0011] The left flank sequence consists of bases 1 to 296 from chromosome 5 of the rice genome and bases 297 to 1241 from the GATV3 vector sequence. The left flank sequence is the 5'-end border flanking sequence of the exogenous insertion vector of the rice GAT transformation event GATV3-867-3.

[0012] The above-mentioned right-wing and left-wing sequences are characteristic sequences of the rice GAT transformation event GATV3-867-3, which can be used to distinguish the rice GAT transformation event GATV3-867-3 from other transgenic / non-transgenic rice, and can be used for qualitative detection and quantitative analysis of the rice GAT transformation event GATV3-867-3.

[0013] Furthermore, the present invention provides primers for detecting the flanking sequences, the nucleotide sequences of the primers are shown in SEQ ID NOs. 11-12 and 9-10.

[0014] Furthermore, the present invention provides the use of the flanking sequence or the primer in detecting or identifying transgenic rice and its derivatives; the transgenic rice is the rice GAT transformation event GATV3-867-3.

[0015] The rice GAT transformation event GATV3-867-3 is a T-DNA fragment of pC0309-KhvMaauMCMK5400 inserted into bases 8873091-8873112 of the non-coding region of chromosome 5 (pC0309-KhvMaauMCMK5400 has been disclosed in Chinese patent application number 202010379287.9).

[0016] Furthermore, the present invention provides a reagent or a kit, which comprises the primers shown in SEQ ID NOs. 11-12 and SEQ ID NOs. 9-10.

[0017] Preferably, the reagent or kit is a reagent or kit for PCR.

[0018] In some embodiments, the reagent or kit further comprises at least one of water, Taq DNA polymerase, dNTPs, PCR buffer, a positive control, and a negative control.

[0019] Furthermore, the present invention provides a method for detecting transgenic rice GATV3-867-3, comprising: detecting whether the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 exist simultaneously in the DNA of a rice sample.

[0020] Preferably, PCR amplification is performed using primers as shown in SEQ ID NOs. 11-12 and 9-10, or reagents or kits containing these primers, with the DNA of the sample to be tested as a template.

[0021] In some embodiments, based on the PCR amplification product, it is determined whether the T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted into bases 8873091-8873112 of the non-coding region of chromosome 5 of the test sample.

[0022] Preferably, if the primer pair shown in SEQ ID NO. 11-12 amplifies a target fragment of 413 bp, and the primer pair shown in SEQ ID NO. 9-10 amplifies a target fragment of 1241 bp, then the sample to be tested contains components derived from GATV3-867-3.

[0023] Preferably, the PCR amplification program includes: 93-95°C for 1-2.5 min; 93-95°C for 20-40 s; 50-60°C for 20-40 s; 70-73°C for 1-1.5 min; 70-73°C for 5-6 min; 23-27°C for 1.5-2.5 min, for 30-35 cycles.

[0024] More preferably, the PCR amplification program includes: 94°C for 2 min; 94°C for 30 s; 55°C for 30 s; 72°C for 1 min; 72°C for 5 min; 25°C for 2 min, for 30-35 cycles.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) This invention discloses for the first time the flanking sequences of the insertion site of the exogenous gene of the rice genetic intelligent breeding technology (GAT) transformation event GATV3-867-3 in the rice genome;

[0027] (2) This invention confirms for the first time the origins of the different bases in the flanking sequences of the insertion site of the exogenous gene in the rice genetic intelligent breeding technology (GAT) transformation event GATV3-867-3 in the rice genome, and determines the junction site sequence where the exogenous vector is inserted into the rice genome sequence;

[0028] (3) Using the flanking sequences discovered in this invention, a specific qualitative detection method for the rice genetic intelligent breeding technology (GAT) transformation event GATV3-867-3 was established for the first time;

[0029] (4) The present invention is applicable to the detection, monitoring and safety management of the rice genetic intelligent breeding technology (GAT) transformation event GATV3-867-3 generation and its derivative lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the phenotype diagram of the rice GAT transformation event GATV3-867-3 sprayed with 5x imazethapyr in Example 1 of the present invention; wherein 0d is before spraying, 14d is 14 days after spraying, WT is the wild type, CK+ is the positive control (imazethapyr-resistant plant), and 867-3 is GATV3-867-3.

[0031] Figure 2 This is a phenotypic diagram of the rice GAT transformation event GATV3-867-3 sprayed with 3 g / L bentazon in Example 1 of the present invention; wherein 0d is before spraying, 7d is 7 days after spraying, 14d is 14 days after spraying, WT is the wild type, CK+ is the positive control (benzon-sensitive mutant), and 867-3 is GATV3-867-3.

[0032] Figure 3 This is a pollen fertility and seed fluorescence diagram of the rice GAT transformation event GATV3-867-3 in Example 1 of the present invention; wherein ZH11 is Zhonghua 11, 867-3 (T0) is the T0 generation of GATV3-867-3, and 867-3 (T1) is the T1 generation of GATV3-867-3.

[0033] Figure 4 This is a hiTail-PCRIII electrophoresis diagram of the right border flanking sequence of the rice GAT transformation event GATV3-867-3 in Example 2 of the present invention; wherein, M is a marker; ddH2O is double-distilled water; ZH11 is the non-transgenic japonica rice Zhonghua 11; 9311 is the non-transgenic indica rice 9311; P is the GATV3 vector plasmid; and 867-3 is the rice GAT transformation event GATV3-867-3.

[0034] Figure 5 Schematic diagram of the integration site of the rice GAT transformation event GATV3-867-3 in the rice genome in Example 3 of the present invention; wherein, the T-DNA of the rice GAT transformation event GATV3-867-3 is inserted into the non-coding region of chromosome 5 of the rice genome at bases 8873091-8873112.

[0035] Figure 6 This is a specific qualitative PCR amplification diagram of the rice GAT transformation event GATV3-867-3 in Example 5 of the present invention; wherein: WT is a non-transgenic rice gene DNA template; 867-3 is a genomic DNA template of the rice GAT transformation event GATV3-867-3. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0037] Where specific techniques or conditions are not specified in the examples, all methods were performed according to conventional methods, techniques or conditions described in literature in the field, or according to product specifications. Reagents and instruments used, for which the manufacturers are not specified, are conventional products that can be purchased through regular channels.

[0038] Example 1 Obtaining Rice GAT Transformation Event GATV3-867-3

[0039] In this example, based on the genetically intelligent breeding technology GAT, pollen fertility restorer genes, pollen abortion genes, herbicide-sensitive genes, and selection marker genes were tightly linked in a specific order and orientation on the vector pC0309-KhvMaauMCMK5400 (GAT) (referred to as the GATV3 vector in this invention). The specific sequence and construction method of the vector pC0309-KhvMaauMCMK5400 were described in reference to Chinese patent application number 202010379287.9, "A genetically intelligent seed breeding system for hybrid crop breeding and its application," and the homozygous recessive male sterility gene was successfully introduced into the rice plant Zhonghua 11 (ZH11). Oscyp704b2-3 GATV3-867-3 is one of the transformation events, which has been disclosed in the conference abstract (The 9th International Conference on Botany, Dec. 8-10, 2023, Characterization of GAT transformation events offered a promising option to GMS application in rice, Xiongxia Jin). The "867-3" mentioned therein refers to this transformation event.

[0040] Based on the various elements of the GAT vector, the transformation event GATV3-867-3 was tested for herbicide phenotype, pollen fertility, and seed fluorescence. The results were as follows: (1) According to the positive / negative herbicide screening, the transformation event GATV3-867-3 showed a high resistance phenotype when sprayed with 5x imidazolinone solution, indicating that the expression cassette of the maintainer screening element worked efficiently and could be used for the purification of the GAT maintainer line. Figure 1When sprayed with 3g / L bentazon solution, the phenotype was highly sensitive, indicating that the herbicide sensitive element expression cassette was highly efficient and sensitive and could be used for purification of GAT sterile lines. Figure 2 (2) Using potassium iodide staining, the fertility of the pollen of the transformation event GATV3-867-3 was tested. The ratio of aborted pollen to fertile pollen was 1:1, indicating that the working efficiency of the restoration gene element and the pollen abortion gene element was high, which enabled the maintenance line to maintain a heterozygous state. Figure 3 (3) According to the results of pollen fertility identification, the self-fertilized seeds will also show a 1:1 separation when observed under a 560-595nm excitation light microscope, that is, 50% of the seeds contain the GAT vector and show deep red fluorescence; 50% of the seeds do not contain the GAT vector and have no fluorescence, indicating that the fluorescent protein expression can work normally and can be used for mechanical seed sorting, see Figure 3 The above functions are present in both T1 and T2, proving that they can be stably inherited from generation to generation.

[0041] In summary, the single-copy transformation event GATV3-867-3, in which all elements function normally, represents an excellent initial maintainer line with genetic stability. Self-pollination of this maintainer line allows for the propagation of sterile and maintainer lines, which can be widely applied to hybrid rice, improving the efficiency of hybrid rice breeding.

[0042] Example 2 Amplification of the Right Border Flanking Sequence of Rice GAT Transformation Event GATV3-867-3

[0043] (1) Extraction of rice genomic DNA using TPS method

[0044] ① Grinding: Take a 3-4 cm long young leaf (1-2 cm long old leaf) of the transgenic rice GAT transformation event GATV3-867-3 obtained in Example 1, place it in a 2 mL centrifuge tube, add 800 μL of TPS extract, add steel balls, and grind for 120 seconds using a cell disruptor (grinding machine);

[0045] ②After grinding, place in a 75℃ water bath and incubate for 30 min;

[0046] ③ Centrifuge at 13000 rpm for 10 min and take the supernatant (about 500 μL) into another 1.5 mL centrifuge tube;

[0047] ④ Add two volumes of pre-cooled anhydrous ethanol or an equal volume of isopropanol, mix gently, and place in a -30°C refrigerator for 2-3 hours (or place in a 4°C refrigerator overnight; or place in a -80°C refrigerator for 1-2 hours) until the DNA precipitates;

[0048] ⑤ Centrifuge at 13,000 rpm for 5 minutes, discard the supernatant, and place the centrifuge tube upside down on the table to air dry;

[0049] ⑥ After air drying, dissolve in 200 μL 1×TE solution or sterile ddH2O; check its integrity by 1.0% agarose gel electrophoresis and determine the DNA concentration by micro-UV spectrophotometer; store in a 4°C refrigerator until ready for use.

[0050] (2) Isolation of T-DNA right flank sequence using hiTail-PCR

[0051] Referring to the high-efficiency thermal asymmetric PCR (hiTail-PCR) method of Liu et al. (2007), three specific primers (GATV3-RB-F1 to F3) were designed based on the right border (RB) sequence of the GATV3 (pC0309-KhvMaauMCMK5400) plasmid map. These primers were combined with the degenerate primers LAD1-1, LAD1-3, and AC1 to isolate the right flank sequence of the T-DNA. The specific primer sequences are shown in Table 1.

[0052] Table 1 hiTail-PCR primers

[0053]

[0054] N is (A / T / G / C), i.e. A or T or G or C; V is (G / C / A), i.e. G or C or A.

[0055] The hiTail-PCR technique was used to amplify the right flanking sequence of the exogenous vector insertion site in the rice GAT transformation event GATV3-867-3 using a three-stage hiTail-PCR reaction. The first hiTail-PCR reaction used two long random primers (LAD1-1 and LAD1-3 mixed in equal proportions) in combination with the specific primer GATV3-RB-F1. Genomic DNA from the rice GAT transformation event GATV3-867-3 was used as a template for PCR amplification. ddH2O, non-transgenic rice lines Zhonghua 11 and 9311, and the GATV3 plasmid served as controls. The product from the first stage PCR amplification reaction was diluted 40-fold and used as a template in the second stage Tail-PCR reaction using the primer combination AC1 / GATV3-RB-F2. The product from the second stage PCR amplification reaction was diluted 10-fold and used as a template in the third stage reaction using the primer combination AC1 / GATV3-RB-F3. The amplified products of the second and third stage reactions were separated by 1.0% agarose gel electrophoresis, and the specific band of 194 bp was selected for sequencing (e.g. Figure 4 ). The PCR reaction system and procedure are shown in Tables 2 and 3.

[0056]

[0057] Example 3 Integration Site of T-DNA of Rice GAT Transformation Event GATV3-867-3 in the Rice Genome

[0058] The specific band PCR product amplified by hiTail-PCR was sequenced to obtain a 194bp right border fusion sequence, as shown in SEQ ID NO. 13. Analysis of the sequence using the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and the GATV3 vector sequence revealed that positions 1 to 95 completely matched the vector right border sequence, and positions 96 to 194 were located in the rice genome sequence and completely matched the published sequence on rice chromosome chr5 (AP014961.1 (8872993 to 8873091). This indicates that the 3' end of the T-DNA of the rice GAT transformation event GATV3-867-3 was inserted into the rice chromosome 5 at base 8873091 (as shown in Figure 1). Figure 5 ).

[0059] Example 4 Amplification of the Left Border Flanking Sequence of Rice GAT Transformation Event GATV3-867-3

[0060] According to the insertion site information obtained in Example 3, a forward primer LB-F2 was designed based on a partial sequence of the vector GATV3, the sequence of which is shown in SEQ ID NO. 9 (5'-GCAATGAATATGCTGCCATCC-3'). A reverse primer G5R1(867-3) was designed based on the published sequence on rice chromosome 5, the sequence of which is shown in SEQ ID NO. 10 (5'-GCCTGATTCTCGCAGTGGTA-3'). This primer pair was used to amplify the left flank sequence of the rice GAT transformation event GATV3-867-3. The amplified product was sequenced, and the resulting sequence was shown in SEQ ID NO. 2, which was 1241 bp in length. Analysis of SEQ ID NO. 2 revealed that the obtained sequence from positions 1 to 296 was located in the rice genome sequence and completely matched the published sequence on rice chromosome chr5 (AP014961.1 (8873112 to 8873407)); positions 297 to 1241 were identical to a partial sequence of the vector GATV3. This means that the 5' end of the T-DNA of the rice GAT transformation event GATV3-867-3 was inserted into the rice chromosome 5 at base 8873112 (e.g., Figure 5 The T-DNA of the rice GAT transformation event GATV3-867-3 was inserted into the rice chromosome 5 at bases 8873091-8873112, resulting in a 20 bp deletion on rice chromosome chr5 (AP014961.1 (8873092 to 8873111), the sequence of which is shown in SEQ ID NO. 14).

[0061] Example 5 Specific PCR Detection Method for Rice GAT Transformation Event GATV3-867-3

[0062] According to Examples 3 and 4, the right and left flanking sequences of the rice GAT transformation event GATV3-867-3 were obtained. Specific primers were designed on rice chromosome chr5 and the exogenous vector, respectively. The specific primer sequences are shown in Table 4. PCR amplification was performed using genomic DNA from the progeny of the rice GAT transformation event GATV3-867-3 as a template and the wild type (WT) as a control. The specific PCR reaction system is shown in Table 5. The amplification program was: 94°C for 2 minutes; 94°C for 30 seconds; 55°C for 30 seconds; 72°C for 1 minute; 72°C for 5 minutes; 25°C for 2 minutes, for 30-35 cycles. PCR products were detected using 1.0% agarose gel (see Table 5). Figure 6 ).

[0063]

[0064] Table 5 Reaction system

[0065]

[0066] The results show that:

[0067] (1) G5F1(867-3) / G5R1(867-3) combination: The rice GAT transformation event GATV3-867-3 template and wild type (WT) can both amplify the 617bp target band (e.g. Figure 6 ) were consistent with expectations and indicated that the rice GAT transformation event GATV3-867-3 was a heterozygous line.

[0068] (2) G5F1(867-3) / RB-R2 combination: The rice GAT transformation event GATV3-867-3 template was able to amplify the 413bp target band of the fusion sequence of the insertion vector and the rice genome at the right border, and the obtained sequence was shown in SEQ ID NO.1, while the wild type (WT) failed to amplify the target band (as shown in Figure 6 ), which was consistent with the expected result, indicating that the rice GAT transformation event GATV3-867-3 contained the GAT vector insert.

[0069] (3) LB-F2 / G5R1(867-3) combination: The rice GAT transformation event GATV3-867-3 template was able to amplify the target band of 1241bp of the fusion sequence of the insertion vector and the rice genome at the left border, and the obtained sequence was shown in SEQ ID NO.2, while the wild type (WT) failed to amplify the target band (as shown in Figure 6), which was consistent with the expected result, indicating that the rice GAT transformation event GATV3-867-3 contained the GAT vector insert.

[0070] The above results indicate that the primer combinations of LB-F2 / G5R1(867-3) and G5F1(867-3) / RB-R2 can amplify the left and right boundary sequences of the rice GAT transformation event GATV3-867-3 and its transformed lines (derivative lines), while no specific fusion bands can be amplified in non-transgenic or other transgenic rice varieties. The above primers can be used to identify the rice GAT transformation event GATV3-867-3 generation and its derivative sequences.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of flanking sequences or primers for detecting the flanking sequences in detecting or identifying transgenic rice and its derivative products; the transgenic rice is the rice GAT transformation event GATV3-867-3; the rice GAT transformation event GATV3-867-3 is a T-DNA fragment inserted into pC0309-KhvMaauMCMK5400 at bases 8873091-8873112 of the non-coding region of chromosome 5; the right flanking sequence of the flanking sequence is shown in SEQ ID NO. 1; the left flanking sequence of the flanking sequence is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The nucleotide sequences of the primers are shown in SEQ ID NOs. 11-12 and 9-10.

3. A method for detecting the rice GAT transformation event GATV3-867-3, characterized in that: include: The rice sample DNA is detected for the simultaneous presence of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2; the rice GAT transformation event GATV3-867-3 is a T-DNA fragment of pC0309-KhvMaauMCMK5400 inserted into bases 8873091-8873112 of the non-coding region of chromosome 5.

4. The method according to claim 3, characterized in that PCR amplification was performed using the primers shown in SEQ ID NOs. 11-12 and 9-10 and the DNA of the sample to be tested as a template.

5. The method according to claim 4, characterized in that Based on the PCR amplification product, determine whether the T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted into the 8873091-8873112 bases of the non-coding region of chromosome 5 of the test sample.

6. The method according to claim 5, characterized in that If the primer pair shown in SEQ ID NOs. 11-12 amplifies a target fragment of 413 bp, and the primer pair shown in SEQ ID NOs. 9-10 amplifies a target fragment of 1241 bp, then the test sample contains the T-DNA fragment component of pC0309-KhvMaauMCMK5400.

7. The method according to any one of claims 4 to 6, characterized in that The PCR amplification program included: 93-95°C for 1-2.5 min; 93-95°C for 20-40 s; 50-60°C for 20-40 s; 70-73°C for 1-1.5 min; 70-73°C for 5-6 min; 23-27°C for 1.5-2.5 min, for 30-35 cycles.

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