A method for identifying and systemically breeding a fine strain of berberis wilsonii against root rot

Abstract

The application discloses a kind of resistance identification and system breeding method of excellent strain of anti-root rot Coptis, it is related to the technical field of medicinal plant disease resistance identification and strain breeding, the application is first according to pathogen composition, inoculation body preparation, seedling age, substrate treatment and environment window formation comparable batch group in seedling inoculation stage, resistance observation record generation group calibration processing result is introduced into the same batch of Coptis control strain, only allow comparable batch group to enter group screening in group calibration processing result is effective state. In this way, the resistance observation record that enters the same comparison chain meets the two prerequisites of consistent inoculation condition and batch response effective simultaneously, batches of inoculation failure, inoculation too strong, control loss or control deviation anomaly no longer directly enter group retention set, so that the disease level source of Coptis candidate strain is constrained to the same inoculation pressure and the same observation prerequisite, solve the problem that the disease level source of batches is inconsistent in front and back batches in multiple batches screening is directly combined and tabled.

Description

Technical Field

[0001] This invention relates to the field of disease resistance identification and strain selection technology of medicinal plants, and in particular to a method for resistance identification and systematic selection of superior strains of Coptis chinensis resistant to root rot. Background Technology

[0002] Coptis chinensis is a perennial medicinal plant, with its rhizome being the medicinal part. Root rot directly affects seedling survival, post-transplant population stability, and the medicinal quality of the rhizome. In the selection and breeding of superior Coptis chinensis varieties resistant to root rot, it is usually necessary to first identify the resistance of candidate materials, and then proceed with seed saving, re-screening, and advancement based on this identification. Current techniques often involve artificial inoculation followed by resistance screening based on disease severity, disease index, or symptom presentation, which can be supplemented by phenotypic observation and microecological testing results.

[0003] When conducting multiple batches of resistance screening in Coptis chinensis seedling greenhouses or mountain propagation sites, changes in pathogen composition, inoculum preparation, seedling age, substrate treatment, and environmental conditions during observation can lead to different infection pressures and disease backgrounds for the same candidate material obtained in different batches. Existing technologies typically reduce bias by standardizing operating procedures, setting controls, or increasing the number of rescreenings. However, they lack stable data access constraints regarding whether data from different batches share the same comparative premises and whether they can enter the same screening judgment chain. This can easily lead to problems such as directly merging observation results from different batches, distorted screening results within groups, repeated switching of rescreening destinations, and seed retention criteria that do not correspond to specific inoculation conditions.

[0004] In areas where Coptis chinensis is co-cultivated, understory, or where different soil sources coexist, soil background, pathogen composition, and beneficial bacteria treatment status all influence plant performance. In such cases, the resistance levels, symptom differences, or related observations obtained from different sites may simultaneously reflect both the host material's own resistance and the environmental influences of the microecological conditions. While existing technologies can reduce bias through multi-site trials, control settings, or additional testing, they lack stratified confirmation constraints that are aligned with the breeding process to ensure that evidence obtained at different microecological levels can be directly used for the same advancement judgment. This can easily lead to materials performing well under specific environmental conditions prematurely entering subsequent populations, resulting in unstable resistance positional relationships and difficulties in maintaining a continuous chain of evidence during propagation or cross-site retesting.

[0005] Therefore, how to establish comparability constraints on batch data entering the same breeding judgment chain under the conditions of multiple batches of Coptis chinensis screening for root rot resistance and multiple microecological conditions, and how to establish stratified confirmation constraints on resistance evidence entering the advancement judgment, has become a technical problem that needs to be solved. Summary of the Invention

[0006] This application provides a method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot, solving the problems of incomparable batch data and difficulty in stably confirming cross-microecological evidence in the breeding of Coptis chinensis strains resistant to root rot.

[0007] This invention provides a method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot, comprising:

[0008] According to the inoculation batches with records of pathogen composition, inoculum preparation, seedling age, substrate treatment and environmental window, multiple candidate lines of Coptis chinensis were inoculated with pathogens, and individual plant identification records, pedigree records, resistance observation records and sample transfer records were obtained;

[0009] The resistance observation records are grouped into comparable batch groups according to the consistency of the inoculation batches. The resistance observation records of the same batch of Coptis chinensis control lines are used to generate group verification results. Only for comparable batch groups whose group verification results are in an effective state, the same batch of Coptis chinensis control lines, disease grade records, survival status records, and root symptom records are combined to form an intra-group retention set. When the same Coptis chinensis candidate line corresponds to multiple comparable batch groups, each of the comparable batch groups is used as an independent comparison unit, and the corresponding intra-group retention results are retained respectively.

[0010] The candidate strains of Coptis chinensis and their control strains within the group are configured in a reference microecological layer and a non-reference microecological layer for stratified confirmation. When any configured microecological layer lacks a confirmation record, it is written into the rescreening queue. In the absence of any missing confirmation records, when the resistance level is consistent, or the resistance level is within the allowable fluctuation range of the resistance level relative to the reference microecological layer, and maintains the same directional level difference relative to the Coptis chinensis control strain in the same layer, and the range of inter-layer level difference does not exceed the upper limit of the allowable range of inter-layer level difference, it is written into the superior strain candidate library. When the resistance level is inconsistent, or the resistance level is consistent but does not maintain the same directional level difference, it is written into the observation queue. The corresponding results are associated with the pedigree record and the sample transfer record to generate seed retention task record, rescreening task record and delayed confirmation task record, respectively.

[0011] In some embodiments, the resistance observation record includes disease severity record, survival status record, and root symptom record collected at the same observation point;

[0012] The intragroup retention set is determined by first comparing disease severity records, then comparing survival status records, and finally comparing root symptom records to form the intragroup resistance level.

[0013] In some embodiments, when forming the comparable batch group, resistance observation records of the same batch of Coptis chinensis control lines are also obtained, and group verification processing results are generated based on the disease grade records, survival status records and root symptom records.

[0014] The group verification results include valid state, weak state, strong state, and invalid state. Among them, the valid state is that the control lines of the same batch of Coptis chinensis form a pre-calibrated effective control response window within the specified observation window and the control deviation does not exceed the upper limit of the allowable limit. The weak state is that the overall disease incidence of the control lines of the same batch of Coptis chinensis is insufficient. The strong state is that the overall disease incidence of the control lines of the same batch of Coptis chinensis is too fast, the symptoms are concentrated and aggravated or the deaths are concentrated. The invalid state is that the control lines of the same batch of Coptis chinensis are missing, their identities are unclear, the observation records are missing, the observation results are inconsistent with the preset scoring direction, or the observation window is interrupted.

[0015] In some embodiments, when the current batch is determined to be in a valid state, the corresponding Coptis chinensis candidate strain enters the group for screening.

[0016] When a batch is determined to be in a weak, strong, or ineffective state, the corresponding Coptis chinensis candidate strain is added to the set to be reviewed. The original inoculation batch identifier and original resistance observation record are maintained until the review is completed, and the batch is stopped from participating in the screening within the current group.

[0017] In some embodiments, when the same Coptis chinensis candidate line corresponds to multiple comparable batch groups, independent intra-group retention results are retained for each of the comparable batch groups, and resistance observation records of different comparable batch groups are not included in the same comparison process, and a rescreening mark is generated for the Coptis chinensis candidate line.

[0018] In some embodiments, the sample transfer record includes root disease grade sample record, fibrous root sample record, rhizome sample record, rhizosphere sample record, and aboveground phenotypic sample record, all bound to the same single plant identification record. Each sample record corresponds to a sampling time, sampling location, and transfer node.

[0019] Before writing the candidate strains of Coptis chinensis into the candidate pool of superior strains, the screening queue, or the observation queue, an identity consistency check is performed on each sample record.

[0020] In some embodiments, the identity consistency verification includes detecting whether there are sampling location conflicts, transfer node conflicts, or batch affiliation conflicts among the sample records corresponding to the same single plant identification record; when any conflict exists, a source tracing verification is performed based on the single plant identification record, source batch identification, sampling time, sampling location, transfer node, and the material association key jointly generated by the single plant identification record and the pedigree record; when a unique correspondence can be restored after the source tracing verification, a correction write-back or rebinding is performed on the corresponding sample record, and the candidate Coptis chinensis strain is restored to participate in the current group screening or stratified confirmation; when a unique correspondence cannot be restored, the corresponding candidate Coptis chinensis strain is written into the isolation verification queue.

[0021] In some embodiments, the reference microecological layer corresponds to a set of test plots with the same soil source and without the introduction of beneficial bacteria treatment, and the non-reference microecological layer includes a disturbing microecological layer and a risky microecological layer;

[0022] The interfering microecological layer corresponds to a set of test plots where beneficial bacteria have been introduced and the soil source is consistent with the reference microecological layer. The risk microecological layer corresponds to a set of test plots where the continuous cropping status or pathogen spectrum is different from the reference microecological layer.

[0023] In some embodiments, when generating the seed retention task record, rescreening task record, or delayed confirmation task record, the seed retention order and propagation order are generated for the Coptis chinensis candidate lines written into the superior line candidate library, the corresponding inoculation batch identifier and microecological layer record are copied for the Coptis chinensis candidate lines written into the rescreening queue, and the current pedigree position of the Coptis chinensis candidate lines written into the observation queue is retained and the next round of stratified confirmation arrangement is generated.

[0024] In some embodiments, the stratified verification uses reserve or propagated seedlings of the same strain that have been identified during the seedling inoculation stage. These reserve or propagated seedlings are associated with the corresponding Coptis chinensis candidate strains through the single-plant identification record and the pedigree record. When there are missing plants or samples in the configured microecological layer, and the reserve or propagated seedlings of the same strain can be continued within the current observation window, the verification continues according to the original microecological layer and the association relationship of the same material is maintained. When it is impossible to continue or the continued seedlings exceed the current observation window, a rescreening mark is generated and written into the rescreening queue.

[0025] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0026] In this invention, during the seedling inoculation stage, comparable batch groups are first formed based on pathogen composition, inoculum preparation, seedling age, substrate treatment, and environmental window. Then, resistance observation records of the same batch of Coptis chinensis control lines are introduced to generate group verification results. Only comparable batch groups with valid group verification results are allowed to enter the in-group screening. In this way, resistance observation records entering the same comparison chain simultaneously meet the two prerequisites of consistent inoculation conditions and valid batch response. Batches with inoculation failure, excessive inoculation, missing controls, or abnormal control shifts are no longer directly entered into the in-group retention set. This constrains the disease severity source of Coptis chinensis candidate lines to the same inoculation pressure and the same observation premise, solving the problem of inconsistent disease severity sources between batches being directly merged in multi-batch screening.

[0027] When multiple comparable batch groups correspond to the same Coptis chinensis candidate line, independent intra-group retention results are retained, and resistance observation records from different comparable batch groups are not included in the same comparison process. In this way, when multiple batches are repeated, differences in vaccination background are no longer masked by cross-group merging. Instead, the differences are retained in the rescreening stage for further verification, so that the advancement judgment corresponds to the specific source of the vaccination batch, and the basis for seed retention can be traced back to the specific vaccination conditions. This reduces the false triggering of advancement and repeated switching of rescreening paths caused by cross-group conflation.

[0028] Furthermore, the candidate Coptis chinensis strains and their control strains, which are concentrated within the group, are stratified and confirmed in both the reference and non-reference microecological layers. They are required to have consistent resistance levels or be within the allowable fluctuation range of resistance levels, maintain a consistent resistance level difference relative to the control strains in the same layer, and ensure that the variation range of resistance level differences between layers does not exceed the upper limit of the allowable variation range. Only then are they included in the candidate pool of superior strains. In this way, materials entering the candidate pool not only exhibit the same resistance level across different microecological layers but also maintain a consistent positional relationship relative to the control strains in the same layer. This helps to distinguish the environmental impacts of site-specific disease suppression, beneficial bacteria treatment status, continuous cropping status, and changes in pathogen spectrum from the stable resistance performance of the Coptis chinensis strains themselves, avoiding the premature inclusion of materials adapted only to specific sites or treatment conditions into the core population.

[0029] Furthermore, the confirmation results are linked to pedigree records and sample transfer records, generating separate records for seed preservation, rescreening, and delayed confirmation. This ensures that each breeding destination corresponds to the individual plant's identity, pedigree origin, inoculation batch origin, and stratified confirmation origin. In this way, subsequent seed preservation, propagation, rescreening, and delayed confirmation are all built upon a continuous chain of evidence, establishing a continuous correspondence between material origin, sample origin, and determination origin. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.

[0031] Figure 1 This is a flowchart illustrating the resistance identification and systematic breeding method of superior Coptis chinensis strains resistant to root rot in the embodiments. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0033] All terms used in this application (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted in a manner consistent with the context of this specification, and not in an idealized or overly rigid way.

[0034] To facilitate understanding, the relevant terms and concepts involved in the embodiments of this application will be introduced below:

[0035] Candidate lines of Coptis chinensis refer to Coptis chinensis materials that have entered the seedling inoculation stage and are awaiting evaluation for resistance to root rot. Control lines of Coptis chinensis refer to Coptis chinensis materials that are simultaneously deployed with candidate lines under the same batch inoculation and stratum confirmation conditions, used to characterize batch effectiveness and serve as a benchmark for intra-stratum comparison. Individual plant identification records refer to data records used to uniquely identify the origin, seedling location, and sampling association of a single plant. Phylogenetic records refer to data records used to characterize the origin, propagation generation, seed-saving destination, and association of candidate lines with reserve seedlings of the same line. Inoculation batches refer to a set of inoculation operation records formed under the same pathogen composition, inoculum preparation, seedling age, substrate treatment, and environmental window conditions. Comparable batch groups refer to a data set consisting of resistance observation records with consistent inoculation batches and observation windows.

[0036] The allowable fluctuation range of resistance level refers to the range of levels in the non-reference micro-ecological layer that are allowed to deviate from the reference micro-ecological layer.

[0037] Specifically, the observation window is uniformly calculated from the time of inoculation completion and recorded in days post-inoculation. The days post-inoculation number field is used to identify the observation time point. Resistance observation records within the same comparable batch group are aligned using the same days post-inoculation number field. Ideally, three observation time points are set, with an adjustable range of two to five. The interval between adjacent observation time points is preferably 3 to 7 days. To ensure consistency in comparisons within the same batch, a batch index key field is added. This batch index key is an index field formed by sequentially piecing together pathogen composition, inoculum preparation, seedling age, substrate treatment, and environmental window information. An observation alignment key field is also added. This observation alignment key is an alignment field formed by combining the batch index key and the days post-inoculation number field. When resistance observation records enter the same comparable batch group, consistency in both the batch index key and the observation alignment key is used. If any record lacks a batch index key or observation alignment key, or if the number of days post-inoculation exceeds the allowable deviation of the current observation window, it will not enter that comparable batch group, and the original resistance observation record and original sample transfer record will be maintained. The allowable deviation of the observation window is the implementation parameter, preferably 0~1 day, which is adjusted according to the observation frequency of the seedling greenhouse or mountain breeding site to avoid cross-batch merging errors caused by recording time drift.

[0038] The group verification result refers to the judgment result formed on whether the current inoculated batch meets the premise of valid comparison based on the resistance observation records of the control lines of the same batch of Coptis chinensis. The reference microecological layer refers to the set of microecological conditions used to support basic confirmation. The non-reference microecological layer refers to the set of microecological conditions used to support environmental disturbance or risk background confirmation. The sample transfer record refers to the data set that records the source relationship and node status of root disease-grade samples, fibrous root samples, rhizome samples, rhizosphere samples, and aboveground phenotypic samples during the sampling, handover, testing, storage, and result writing process. The intragroup retention set refers to the set of Coptis chinensis candidate lines retained after resistance level ranking within comparable batch groups. The superior line candidate pool refers to the set of Coptis chinensis candidate lines that simultaneously meet the intragroup retention result and cross-layer confirmation result. The rescreening cohort refers to the set of Coptis chinensis candidate lines that enter the next round of inoculation or confirmation due to batch verification, missing confirmation, or incomplete result closure. The observation cohort refers to the set of Coptis chinensis candidate lines whose performance fluctuates but still retains their pedigree position.

[0039] Example 1:

[0040] like Figure 1 As shown, this embodiment presents a method for resistance identification and systematic breeding of superior Coptis chinensis varieties resistant to root rot. Through continuous processing of inoculation batch constraints, intragroup screening, stratified confirmation, and task output, a closed-loop process is formed from seedling inoculation to seed saving, re-screening, and delayed confirmation, specifically including:

[0041] Step S1: According to the inoculation batches that record the pathogen composition, inoculum preparation, seedling age, substrate treatment and environmental window, multiple Coptis chinensis candidate lines are inoculated with pathogens, and single plant identification records, pedigree records, resistance observation records and sample transfer records are obtained;

[0042] Step S2: Group the resistance observation records according to the consistency of the inoculation batches to form comparable batch groups. Obtain the resistance observation records of the control line of the same batch of Coptis chinensis to generate group verification results. Only for comparable batch groups with effective group verification results, combine the control line of the same batch of Coptis chinensis with disease grade records, survival status records, and root symptom records to form an intra-group retention set. When the same Coptis chinensis candidate line corresponds to multiple comparable batch groups, retain independent intra-group retention results for each comparable batch group, and do not include the resistance observation records of different comparable batch groups in the same comparison process.

[0043] Step S3: The candidate strains of Coptis chinensis and their control strains within the group are configured in the reference microecological layer and non-reference microecological layer for stratified confirmation. When any configured microecological layer lacks a confirmation record, the candidate strain of Coptis chinensis is added to the rescreening queue. In the absence of a missing confirmation record, when the resistance level in each effective non-reference microecological layer is consistent with that of the reference microecological layer, or is within the allowable fluctuation range of the resistance level relative to the reference microecological layer, and maintains the same directional level difference relative to the control strain of Coptis chinensis in the same layer, and the range of interlayer level difference does not exceed the upper limit of the allowable range of interlayer level difference, the candidate strain of Coptis chinensis is added to the superior strain candidate library. When the aforementioned conditions for adding to the superior strain candidate library are not met, the candidate strain of Coptis chinensis is added to the observation queue. When comparing discrete levels directly, the allowable fluctuation range is 0 to 1 level unit; or it is calibrated according to historical effective confirmation records, not exceeding the preset adjacent level range.

[0044] Step S4: Associate the corresponding results with the pedigree record and sample transfer record; generate seed retention task records for Coptis chinensis candidate lines written into the superior line candidate library, generate re-screening task records for Coptis chinensis candidate lines written into the re-screening queue, and generate delayed confirmation task records for Coptis chinensis candidate lines written into the observation queue.

[0045] During the seedling inoculation stage, a single-plant identification record is established for each Coptis chinensis seedling, and the corresponding pedigree record is associated with the single-plant identification record and written into the pedigree record. The pathogen composition in the inoculation batch is used to record the types of pathogens involved in the inoculation and their combinations; inoculum preparation is used to record the inoculum source, activation source, and preparation time; seedling age is used to record the growth stage of the Coptis chinensis seedling at the time of inoculation; substrate treatment is used to record the source of the seedling substrate, pretreatment state, and moisture content; and the environmental window is used to record the temperature, humidity, and shading conditions during the specified observation period after inoculation. Resistance observation records consist of multi-timepoint disease severity records, survival status records, and root symptom records within a fixed observation window. When forming comparable batch groups, only resistance observation records with consistent pathogen composition, inoculum preparation, seedling age, substrate treatment, and environmental window, and with consistent observation windows, are included in the same comparable batch group. Resistance observation records with missing, conflicting, or abnormal result rewrites are not included in that comparable batch group. After the intra-group screening is completed, the candidate strains of Coptis chinensis corresponding to the intra-group retention set are moved into the stratified confirmation stage, and the seed retention task record, re-screening task record or delayed confirmation task record are output according to the association status of intra-group retention results, cross-stratified confirmation results and sample transfer records.

[0046] For example, the single-plant identification record includes at least the single-plant source number, seedling location number, and sampling association number; the pedigree record includes at least the strain source, propagation generation, retention destination, and association fields with reserve seedlings of the same strain; and the sample transfer record includes at least the sample type, source single-plant identifier, source batch identifier, sampling time, sampling location, transfer node, and result write-back status. To ensure continuous traceability of the same Coptis chinensis candidate strain between the seedling inoculation stage and the stratified confirmation stage, a material association key field is added. The material association key is an association field jointly generated by the single-plant identification record and the pedigree record, and is synchronously written in the resistance observation record, confirmation record, seed saving task record, rescreening task record, and delayed confirmation task record. When generating each task record, the inoculation batch identifier, observation window identifier, microecological layer identifier, and result generation time are also synchronously written to ensure that subsequent seed saving, rescreening, and delayed confirmation can correspond to the same batch source, the same observation window, and the same microecological layer source.

[0047] Confirmation records must include at least the microecological layer identifier, plot identifier, observation time point, disease level record, survival status record, root symptom record, and the observation results corresponding to the control strain in the same layer. Consistent resistance level means that the same candidate strain of Coptis chinensis falls into the same resistance level in both the reference microecological layer and each effective non-reference microecological layer. Maintaining a consistent resistance level relative to the control strain in the same layer means that the candidate strain of Coptis chinensis maintains a resistance level relationship of higher, equal to, or lower than the control strain in the same layer in both the reference microecological layer and each effective non-reference microecological layer. If any essential microecological layer has a missing plant, the sample and corresponding candidate strain cannot be consistently matched, the control strain in the same layer is missing, or the observation window is interrupted, the corresponding confirmation record is recorded as a missing confirmation record, and the candidate strain of Coptis chinensis is added to the rescreening queue.

[0048] The essential microecological layer refers to the reference microecological layer and at least one non-reference microecological layer that have been actually configured for the same Coptis chinensis candidate line in the current stratified confirmation arrangement. The effective non-reference microecological layer refers to the non-reference microecological layer that has been planted, has a complete observation window, and has complete observation records for the Coptis chinensis control line in the same layer. During stratified confirmation, the same Coptis chinensis candidate line and its control line adopt synchronous planting and synchronous observation in each essential microecological layer. It is preferred to control the planting date difference between each layer within 0-3 days. The observation time point is recorded using the same post-inoculation or post-planting digital segment as the reference microecological layer. The post-planting digital segment is a time field used to identify the observation time point of stratified confirmation, so as to reduce the grade drift caused by phenological period differences. When a missing plant is found, first check the single plant identification record, pedigree record and sample transfer record corresponding to the missing plant; if it can be continued by a reserve seedling of the same strain and the continuation time still falls within the current observation window, continue to confirm according to the original microecological layer and maintain the same material association key; if it cannot be continued or exceeds the current observation window after continuation, record the microecological layer as a missing confirmation record.

[0049] In one implementation of Example 1, the resistance observation record includes disease severity record, survival status record, and root symptom record collected at the same observation point;

[0050] The within-group retention set is determined by first comparing disease severity records, then survival status records, and finally root symptom records to form the within-group resistance level;

[0051] Disease severity records are based on the distribution of root rot symptoms in the rhizome, fibrous roots, and root surface tissues, and include at least the extent of rot, degree of browning, tissue continuity, and the state of fibrous root retention. Survival status records are used to identify normal survival, endangered survival, and death. Root symptom records are used to document rhizome softening, root surface peeling, off-odor, reduction of absorbing roots, and the state of new root regeneration. In the formation of intragroup resistance grades, the initial priority is first determined based on disease severity records at the same observation point; then, the initial priority is adjusted based on survival status records; finally, materials with the same priority are distinguished based on root symptom records, thus obtaining the intragroup retention set.

[0052] In one implementation of Example 1, when the same Coptis chinensis candidate line corresponds to multiple comparable batch groups, independent intra-group retention results are retained for each comparable batch group, and resistance observation records of different comparable batch groups are not included in the same comparison process, and a rescreening mark is generated for the Coptis chinensis candidate line.

[0053] For the same Coptis chinensis candidate line in different comparable batch groups, the original inoculation batch identifier, intra-group resistance grade, status of the same batch of Coptis chinensis control line, and observation window source are retained respectively. Cross-group averaging, cross-group summation, or cross-group merging are not performed. The rescreening markers should include at least the Coptis chinensis candidate line identifier, the identifiers of multiple associated comparable batch groups, the intra-group resistance grade of each group, and the inoculation condition items to be replicated for the next round of rescreening. During rescreening, the inoculation condition items that caused differences in the previous round are replicated according to the rescreening markers, and new inoculation batches are generated for comparison again.

[0054] In one implementation of Example 1, the sample transfer record includes root disease grade sample record, fibrous root sample record, rhizome sample record, rhizosphere sample record and aboveground phenotypic sample record, which are bound to the same single plant identification record. Each sample record corresponds to the sampling time, sampling location and transfer node.

[0055] Before adding Coptis chinensis candidate lines to the superior line candidate library, re-screening queue, or observation queue, perform identity consistency verification on each sample record.

[0056] Each sample record must include at least the sample type, source plant identifier, source batch identifier, sampling time, sampling location, transfer node, and result write-back status. Root disease grade sample records are used to bind the root sampling entity corresponding to the disease grade determination. Fibrous root sample records are used to bind fibrous root lesions and regeneration status. Rhizome sample records are used to bind rhizome tissue symptoms and medicinal quality testing source. Rhizosphere sample records are used to bind rhizosphere microecological testing source. Aboveground phenotypic sample records are used to bind petiole, leaf, and plant shape status. During identity consistency verification, the source plant identifier and source batch identifier are used as the primary association fields, and the sampling time, sampling location, and transfer node are used as secondary association fields. Each sample record corresponding to the same Coptis chinensis candidate line is checked item by item.

[0057] It is understood that sampling time is recorded using a unified clock and accurate to the minute, and sampling location is recorded using at least one of the following: seedling location number, plot location number, or sampling part number. Transfer nodes are recorded in the order of sampling, handover, testing, storage, and result write-back. To reduce errors and conflicts caused by manual data entry differences, the allowable deviation for sampling time is the implementation parameter, preferably 0-30 minutes. Root disease-grade sample records, fibrous root sample records, and rhizome sample records from the same source at the same observation time should share the same source batch identifier. Rhizosphere sample records are allowed to have a 0-1 observation time misalignment with aboveground phenotypic sample records, but the same material association key should be maintained. When conflicts occur in sampling location, transfer node, or batch affiliation, the original records should be kept unchanged, and a conflict source field and a verification time field should be added to the sample transfer record. After verification, only the verified sample records are written back; sample records that fail verification remain isolated and do not participate in the current group screening and stratified confirmation.

[0058] In one implementation of Example 1, identity consistency verification includes detecting whether there are sampling location conflicts, transfer node conflicts, or batch affiliation conflicts among sample records corresponding to the same single plant identification record.

[0059] When any conflict exists, traceability verification is performed based on the single plant identification record, source batch identification, sampling time, sampling location, transfer node, and material association key. If a unique correspondence can be restored after traceability verification, the corresponding sample record is corrected and rebound, and the candidate Coptis chinensis strain is restored to participate in the current group screening or stratified confirmation. If a unique correspondence cannot be restored, the corresponding candidate Coptis chinensis strain is written into the isolation verification queue.

[0060] Sampling location conflict refers to a single plant from the same source being recorded as two incompatible sampling sites or two incompatible plots at the same sampling time. Transfer node conflict refers to the same sample simultaneously corresponding to multiple mutually exclusive transfer nodes in time sequence, or a node reversal state where sampling precedes testing or testing precedes writing. Batch attribution conflict refers to the same sample being simultaneously associated with two different inoculation batches or two different microecological levels. The original pedigree records of Coptis chinensis candidate lines in the isolation and verification queue remain unchanged, and their superior line candidate library writing status is frozen. Once the conflict is resolved, the intra-group screening or stratified confirmation steps before the interruption will be restored.

[0061] In one implementation of Example 1, the reference micro-ecological layer corresponds to a set of test plots with the same soil source and without the introduction of beneficial bacteria treatment, and the non-reference micro-ecological layer includes the disturbing micro-ecological layer and the risk micro-ecological layer.

[0062] The disturbing microecological layer corresponds to the set of test plots that have been treated with beneficial bacteria and whose soil source is the same as that of the reference microecological layer; the risk microecological layer corresponds to the set of test plots that have different continuous cropping status or pathogen spectrum from the reference microecological layer.

[0063] Soil source is used to record the collection, mixing, and pretreatment sources of the soil for seedling or cultivation. Continuous cropping status is used to record whether the target plot has been continuously planted with Coptis chinensis or related hosts. Pathogen spectrum is used to record the composition of major pathogens detected in the target plot or rhizosphere samples. Beneficial microbial treatment status is used to record whether beneficial microbial treatment was applied before inoculation, before planting, or after planting. Plots in the reference microecological layer maintain consistent soil sources, do not introduce beneficial microbial treatments, and are used as the basic confirmatory environment. Plots in the interfering microecological layer maintain consistent soil sources with the reference microecological layer but introduce beneficial microbial treatments. Plots in the risk microecological layer differ from the reference microecological layer at least in terms of continuous cropping status or pathogen spectrum. The same Coptis chinensis candidate line and its control line are confirmed using a paired deployment method across different microecological layers.

[0064] When pairing and deploying, the planting quantity of the same Coptis chinensis candidate line in the reference microecological layer, disturbance microecological layer, and risk microecological layer should be the same, preferably maintaining an equal number of lines in each layer. The Coptis chinensis control line should be synchronously deployed in each layer and should maintain the same planting date and observation window as the corresponding Coptis chinensis candidate line. The consistency of soil source is determined by the simultaneous consistency of the records of soil collection source, mixing source, and pretreatment source. The pathogen spectrum can be obtained based on existing detection records of plot samples or rhizosphere samples, as long as the detection standards are consistent within the same breeding cycle. When a plot experiences sudden waterlogging, continuous abnormal temperature and humidity fluctuations, or failure of beneficial bacteria treatment, the existing microecological layer identifier should remain unchanged, and the abnormal operating condition field should be written into the confirmation record. The abnormal operating condition field is used to record the source of environmental abnormalities and does not participate in the resistance level calculation, but it serves as the basis for determining whether to include missing confirmation records and whether to continue using the stricter threshold standard from the previous round.

[0065] In one implementation of Example 1, when generating seed retention task records, rescreening task records, or delayed confirmation task records, the seed retention order and propagation order are generated for the Coptis chinensis candidate lines written into the superior line candidate library, the corresponding inoculation batch identifier and microecological layer record are copied for the Coptis chinensis candidate lines written into the rescreening queue, and the current pedigree position of the Coptis chinensis candidate lines written into the observation queue is retained and the next round of stratified confirmation arrangement is generated.

[0066] The seed preservation task record should include at least the candidate strain identifier, pedigree location, source of seed preservation, batch of inoculated strain, microecological layer passed through, and propagation destination. The rescreening task record should include at least the candidate strain identifier, batch of inoculated strain to be re-verified, microecological layer to be re-verified, type of sample to be collected, and next round of observation window. The delayed confirmation task record should include at least the candidate strain identifier, current pedigree location, reason for delay, source of evidence to be preserved, and next round of confirmation plot. The candidate strains of Coptis chinensis included in the superior strain candidate pool should be arranged in seed preservation and propagation order according to pedigree continuity and cross-layer confirmation stability; the candidate strains of Coptis chinensis included in the rescreening or observation queue should maintain their original pedigree location and retain all historical transfer records.

[0067] In one implementation of Example 1, the hierarchical verification uses the same lineage of reserve seedlings or propagation seedlings that have completed identity binding during the seedling inoculation stage. The same lineage of reserve seedlings or propagation seedlings are associated with the corresponding Coptis chinensis candidate lineage through single plant identification records and pedigree records.

[0068] "Reserve seedlings of the same strain" refers to reserve seedlings that were developed simultaneously with the target Coptis chinensis candidate strain during the seedling inoculation stage, were not used for destructive sampling, and have already been identified. "Propagated seedlings" refers to Coptis chinensis materials obtained from the candidate strains that have entered the subsequent confirmation stage through seed saving or propagation, and that maintain a continuous association with the original pedigree record. During stratified confirmation, reserve seedlings of the same strain are used first; if reserve seedlings of the same strain are insufficient, propagated seedlings are used. During the process of using these seedlings, the source identifier, pedigree source, corresponding inoculation batch, and corresponding microecological layer of the reserve seedlings or propagated seedlings of the same strain are simultaneously written into the pedigree record and sample transfer record.

[0069] Before reserve or propagated seedlings of the same strain enter stratified confirmation, their individual plant identification record, pedigree record, material association key, and source batch identification are verified. If all four are consistent, they maintain the same strain origin and pedigree position as the target Coptis chinensis candidate strain; otherwise, they do not enter the current stratified confirmation. When using propagated seedlings, the propagation source, propagation batch, and propagation completion time are also recorded, and the same material association key as the original Coptis chinensis candidate strain is maintained to ensure the continuity of the evidence chain across stages. If the number of reserve seedlings and propagated seedlings is insufficient to cover all necessary microecological layers, priority is given to ensuring that the reference microecological layer and at least one non-reference microecological layer are configured. The remaining unconfigured microecological layers do not enter the current round of missing data determination. Microecological layers that have been configured but did not complete the observation window due to material loss during the process are still recorded as missing data confirmation records.

[0070] In a preferred implementation of Example 1, when the resistance level of the same Coptis chinensis candidate line is determined to maintain the same directional grade difference relative to the control line in the same layer, the resistance level of the same Coptis chinensis candidate line in the reference microecological layer, the interfering microecological layer and the risk microecological layer is uniformly converted into a grade order value arranged in order of resistance from weakest to strongest, and then compared with the control line in the same layer within each layer.

[0071] The difference in grade within a layer is expressed as:

[0072] ,

[0073] in, Candidate strain of Coptis chinensis In the micro-ecological layer The difference in grade within the same layer compared to the control strain of Coptis chinensis; Candidate strain of Coptis chinensis In the micro-ecological layer The resistance level sequence value in the text; For the same micro-ecological layer Resistance rank values ​​of the control strain of Coptis chinensis; The microecological layer is identified by the reference microecological layer, the disturbing microecological layer, or the risk microecological layer. The resistance grade sequence value is assigned according to a unified grade table within the same breeding cycle. The higher the grade, the stronger the resistance. Therefore, a grade difference greater than 0 within a layer indicates that the candidate Coptis chinensis line is higher than the control Coptis chinensis line in the same layer, a grade difference less than 0 within a layer indicates that the candidate Coptis chinensis line is lower than the control Coptis chinensis line in the same layer, and a grade difference of 0 within a layer indicates that the two are at the same grade.

[0074] To avoid misjudgment of direction due to single-level boundary fluctuations, intra-layer direction states are generated based on intra-layer level differences and used as the direct basis for determining same-direction level differences. Intra-layer direction states are represented as follows:

[0075] ,

[0076] in, Candidate strain of Coptis chinensis In the micro-ecological layer The intralayer orientation state; For the micro-ecological layer The orientation determination threshold is used to suppress minor fluctuations near grade boundaries. When discrete grades are directly compared using integer grades, the orientation determination threshold is set to 1 grade unit. When the grade order value is calculated by comprehensively converting disease grade records, survival status records, and root symptom records, the orientation determination threshold is set to half the median distance between the centers of adjacent grades, and is limited to between 0.5 and 1 grade unit. An intra-layer orientation state of 1 indicates that the candidate Coptis chinensis strain is higher than the control Coptis chinensis strain in that layer; an intra-layer orientation state of 0 indicates that the candidate Coptis chinensis strain and the control Coptis chinensis strain in that layer have no significant orientation difference; an intra-layer orientation state of... When the time is right, it indicates that the candidate strain of Coptis chinensis is lower than the control strain of Coptis chinensis in that layer.

[0077] Based on this, when the reference microecological layer has a confirmed record, and at least one of the interfering microecological layer and the risk microecological layer has a valid confirmed record, the intra-layer directional state of the reference microecological layer is read as the reference direction; the layers with valid confirmed records in the interfering microecological layer and the risk microecological layer are compared with the reference direction respectively; when only one of the interfering microecological layer and the risk microecological layer has a valid confirmed record, that layer is used as the source of direction determination for the non-reference microecological layer; when both layers have valid confirmed records, the candidate Coptis chinensis strain is considered to maintain the same directional grade difference only when the intra-layer directional state of both layers is consistent with the reference direction.

[0078] To limit the situation where the direction is consistent but the amplitude drift between layers is too large, the range of variation of the grade difference within each layer is also compared. The range of variation is expressed as follows:

[0079] ,

[0080] in, Candidate strain of Coptis chinensis The range of variation in the inter-level grade difference; Candidate strain of Coptis chinensis Effective confirmation of the microecological layer collection; To effectively confirm the maximum value of the hierarchical difference within the microecological layer set; To effectively confirm the minimum value of the hierarchical difference within the microecological layer set.

[0081] The range of interlayer grade difference variation is calculated only when the set of validly confirmed micro-ecological layers simultaneously includes a reference micro-ecological layer and at least one non-reference micro-ecological layer; when this condition is not met, the range of interlayer grade difference variation is not generated, and the corresponding situation is regarded as a missing confirmed record.

[0082] The same-direction grade difference includes same-direction advantage, same-direction disadvantage, and no significant directional difference in the same direction. When the intra-layer directional state of the reference microecological layer and each effective non-reference microecological layer is the same, it is considered that the same-direction grade difference is maintained. When the intra-layer directional state of the reference microecological layer and any effective non-reference microecological layer is different, it is considered that the directions are inconsistent. The upper limit of the allowable range of inter-layer grade difference variation is determined according to the historical valid confirmed records. When comparing discrete grades directly, one grade unit is taken. When converting the grade ordinal value of the comprehensive observation value, the upper quartile value of the range of inter-layer grade difference variation in the historical valid confirmed records is taken, and it is limited to no more than two grade units. Historical valid confirmed records refer to the set of historical confirmed records where the confirmed records of the reference microecological layer and at least one non-reference microecological layer are complete, the control strain of Coptis chinensis in the same layer has formed a valid direction determination, and the cross-layer destination determination has been completed.

[0083] Therefore, when performing cross-strata confirmation, it is first determined whether the resistance grade sequence values ​​of the same Coptis chinensis candidate line in each effective non-reference microecological layer are consistent with the resistance grade sequence values ​​in the reference microecological layer, and then it is determined whether it maintains the same grade difference relative to the Coptis chinensis control line in the same layer. The necessary microecological layer refers to the reference microecological layer and non-reference microecological layer that have been configured for the Coptis chinensis candidate line in the current stratified confirmation arrangement; microecological layers not configured in this round of stratified confirmation arrangement are not considered as objects of judgment for missing confirmation records.

[0084] When both conditions are met, the candidate Coptis chinensis strain is deemed to have a stable resistance positional relationship in different microecological layers and is added to the candidate pool of superior strains. When any essential microecological layer lacks a confirmed record, or although there is an observation record, the intra-layer directional state cannot be generated because the Coptis chinensis control strain in the same layer has not formed an effective directional determination, it does not enter the directional conflict determination and is directly added to the re-screening queue. When the confirmed records of each essential microecological layer are complete, but the intra-layer directional state of any effective non-reference microecological layer is inconsistent with that of the reference microecological layer, or the range of inter-layer grade difference exceeds the upper limit of the allowable range of inter-layer grade difference, or the resistance grade sequence value in each effective non-reference microecological layer is inconsistent with that of the reference microecological layer, the candidate Coptis chinensis strain is added to the observation queue.

[0085] A new field, "Version Number for Confirmation Judgment," is added. This field is only used to bind the directional judgment threshold, the upper limit of the allowable range of inter-layer grade difference variation, and the corresponding set of historically valid confirmation records used by the same Coptis chinensis candidate line in a single round of cross-layer confirmation. It is not included in the calculation. The same confirmation judgment version number should be used for the same Coptis chinensis candidate line in the reference microecological layer, the disturbing microecological layer, and the risk microecological layer in this round of cross-layer confirmation. Before the completion of this round of cross-layer confirmation, the versions of the directional judgment threshold and the upper limit of the allowable range of inter-layer grade difference variation will not be switched. The directional judgment threshold and the upper limit of the allowable range of inter-layer grade difference variation will be implemented with a fixed standard within the same breeding cycle. When the number of newly added valid confirmation batches reaches the preset number, the threshold will be updated using a sliding window. In the event of sudden waterlogging in continuously cropped plots, incomplete stabilization of soil source replacement, or failure of beneficial bacteria treatment, the threshold update will be suspended, and the stricter directional judgment threshold and the upper limit of the allowable range of inter-layer grade difference variation from the previous round will be used.

[0086] Example 2:

[0087] Based on Example 1, this example provides an implementation method for generating the verification results of the control strains from the same batch of Coptis chinensis. By jointly determining the control response value and the control offset value, it distinguishes between batches that can enter the formal comparison and batches that need to be verified, as follows:

[0088] When forming comparable batch groups, resistance observation records of the same batch of Coptis chinensis control lines are also obtained, and group verification results are generated based on disease grade records, survival status records and root symptom records.

[0089] The group verification results include valid state, weak state, strong state and invalid state. Among them, the valid state is that the control line of the same batch of Coptis chinensis forms a pre-calibrated effective window of control response within the specified observation window and the control deviation does not exceed the upper limit of the allowable limit. The weak state is that the overall disease incidence of the control line of the same batch of Coptis chinensis is insufficient. The strong state is that the overall disease incidence of the control line of the same batch of Coptis chinensis is too fast, the symptoms are concentrated and aggravated or the deaths are concentrated. The invalid state is that the control line of the same batch of Coptis chinensis is missing, the identity is unclear, the observation record is missing, the observation result is inconsistent with the preset scoring direction or the observation window is interrupted.

[0090] In one optional implementation, when the current batch is determined to be in a valid state, the corresponding Coptis chinensis candidate strain enters the group for screening;

[0091] When the current batch is determined to be in a weak state, a strong state, or an ineffective state, the corresponding Coptis chinensis candidate line is written into the set to be reviewed, and the original inoculation batch identifier and original resistance observation record are maintained until the review is completed, and its participation in the current group screening is stopped.

[0092] The interpretation of the group verification results includes four states: valid, weak, strong, and invalid. A valid state means that the control strain in the same batch of Coptis chinensis forms a pre-calibrated valid control response window within the specified observation window, and the control deviation does not exceed the allowable upper limit, thus enabling the current batch to enter the formal comparison. A weak state means that the overall disease incidence in the control strain in the same batch of Coptis chinensis is insufficient, and the control response value is below the lower limit of the valid window, resulting in the current batch not forming sufficient resistance differentiation. A strong state means that the overall disease incidence in the control strain in the same batch of Coptis chinensis is too rapid, symptoms are concentrated and aggravated, or there is concentrated death, causing the control response value to exceed the upper limit of the valid window, or although it is within the valid window, the control deviation exceeds the allowable upper limit, resulting in the current batch not reflecting normal differences. An invalid state means that the control strain in the same batch of Coptis chinensis is missing, its identity is unclear, observation records are incomplete, the observation results are inconsistent with the preset scoring direction, or the observation window is interrupted, resulting in the current batch not being able to form a valid comparison. When the current batch is determined to be in a valid state, the corresponding Coptis chinensis candidate line enters the screening group; when the current batch is determined to be in a weak state, a strong state, or an invalid state, the corresponding Coptis chinensis candidate line is written into the set to be reviewed, and the original inoculation batch identifier and the original resistance observation record are maintained until the review is completed.

[0093] In a preferred implementation of Example 2, the group verification processing result is generated as follows:

[0094] When generating the group verification results, the disease severity records, survival status records, and root symptom records of the same batch of Coptis chinensis control strains were read at the same observation time point and converted into batch-level control response values. The conversion method was as follows:

[0095] ,

[0096] in, For the first Control response values ​​for comparable batch groups; The median value of the disease grade records of the same batch of Coptis chinensis control strains at the same observation time point; The average survival rate of the same batch of Coptis chinensis control strains at the same observation point is recorded as 1 for survival and 0 for death. The median value of root symptoms recorded for the same batch of Coptis chinensis control strains at the same observation time point; , and The intra-batch composite weights for disease severity, survival status, and root symptoms are respectively satisfied. All values ​​are non-negative. The control response value increases with increasing disease severity, mortality rate, and root symptoms, with a lower limit of 0. Disease severity records and root symptom records should use a pre-fixed grading standard within the same breeding cycle and be consistent with the survival status record in direction before being included in the control response value calculation; intra-batch composite weights are only valid under the corresponding scoring standard and observation time point, and should not be reused across different scoring standards, observation time points, or inoculation conditions. When there is an adjustment to the disease severity scoring standard, root symptom grading, or observation time point, the original verification standard version should be frozen, and the statistical values, weights, and thresholds of the old and new versions should not be mixed.

[0097] Furthermore, before being included in the control response value calculation, disease grade records and root symptom records adopt a fixed grade caliber within the same breeding cycle, preferably using a discrete grade of 1-5 or 1-9. Survival status records are uniformly converted to a 0-1 range for both survival and mortality before being included in the synthesis, ensuring consistency in the direction of each observation item. The intra-batch synthesis weights are implementation parameters, preferably with a weight of 0.5 for disease grade records, 0.3 for survival status records, and 0.2 for root symptom records, with adjustable ranges of 0.4-0.6, 0.2-0.4, and 0.1-0.3 respectively, and a total of 1. The offset synthesis weights preferably have a weight of 0.4 for disease grade records, 0.3 for survival status records, and 0.3 for root symptom records, with adjustable ranges of 0.3-0.5, 0.2-0.4, and 0.2-0.4 respectively, and a total of 1. When using a weighted approach, disease severity records play a primary distinguishing role, survival status records identify extreme disease incidence, and root symptom records subdivide boundary materials, thus avoiding misjudgments of weak or strong states due to reliance on a single observation item. The minimum number of control plants is a parameter, preferably no less than 6, with an adjustable range of 6–20. If the number is lower than the minimum control number, no control response value or control offset value is generated; the original inoculation batch identifier is directly retained and written into the set to be reviewed.

[0098] When a batch of Coptis chinensis control lines contains multiple control sources, the corresponding statistical values ​​are first calculated for each control source. Then, the statistical values ​​from each source are weighted and synthesized according to the preset control weights to form the disease severity statistics, survival status statistics, and root symptom statistics used to calculate the control response values. The preset control weights remain unchanged within the same breeding cycle. When a batch of Coptis chinensis control lines contains multiple control sources, the historical reference statistical values ​​used for comparison of the current batch should use the same control source composition and the same preset control weights. If the control source composition or preset control weights change, the statistical values ​​before and after the change should not be directly mixed; the new calibration version should be switched to. If historical reference statistical values ​​of the same version are missing, batch offset recalibration should be performed.

[0099] After obtaining the control response value, it is compared with the historical effective batch reference statistical value under the same pathogen composition, inoculum preparation, seedling age, substrate treatment and environmental window conditions to identify whether the inoculation pressure falls within the effective window and to determine whether there is an abnormal control shift.

[0100] The offset value is represented as follows:

[0101] ,

[0102] in, For the first The control offset values ​​for each comparable batch group; , and These are reference statistical values ​​for the disease grade record, survival status record, and root symptom record of the Coptis chinensis control strain among historical valid batches that have the same pathogen composition, inoculum preparation, seedling age, substrate treatment, and environmental window as the inoculated batch. , and These are the offset synthesis weights, satisfying... All values ​​are non-negative. The larger the control offset value, the more significant the structural deviation of the current batch relative to historical valid batches, with a lower limit of 0. If any of the disease severity record, survival status record, or root symptom record of the same batch of Coptis chinensis control strain is missing at the same observation point, or if the corresponding sample size is lower than the preset minimum control plant number, or if there are abnormal records exceeding the allowable range of the current scoring caliber, the control response value or control offset value will not be calculated, the current batch will not be included in the pass / fail judgment, will be directly written into the pending review set, and an observation anomaly flag will be attached. The observation anomaly flag is only used to record the source of the anomaly and trigger rollback, and is not included in the calculation.

[0103] Based on this, the group verification processing result is determined according to the control response value and control offset value. The group verification processing result includes valid state, weak state, strong state, and invalid state; and a batch processing identifier is further generated, which is represented as:

[0104] ,

[0105] in, For the first Batch processing identifier for comparable batch groups; Results of a repeat test for a failed vaccination; The passing results are required to be included in the formal comparison; For batch offset recalibration results; and These are the lower and upper limits of the effective window for the control response value, respectively. This represents the upper limit of the allowable control offset value. When the control response value is lower than the lower limit of the effective window, it indicates that the pathogen inoculation did not induce sufficient response in the control strain. The Coptis chinensis candidate system I corresponding to the current batch is added to the verification set and a failed inoculation re-calibration flag is attached. When the control response value is within the effective window and the control offset value does not exceed the upper limit of the allowable value, it indicates that the inoculation was successful, the inoculation pressure is within the effective range, and the control did not show abnormal offset. The batch is allowed to enter the formal comparison. When the control response value exceeds the upper limit of the effective window, or although it is within the effective window, the control offset value exceeds the upper limit of the allowable value, it indicates that the batch has excessive inoculation pressure or batch structure offset. The Coptis chinensis candidate system I corresponding to the current batch is added to the verification set and a batch offset recalibration flag is attached. It is then stopped from participating in the current group screening.

[0106] The effective window lower limit, effective window upper limit, and allowable upper limit are calibrated using historical effective batches. The effective window lower limit is used to constrain insufficient vaccination, the effective window upper limit is used to constrain excessive vaccination, and the allowable upper limit is used to constrain control structure deviation.

[0107] The historical valid batch reference set refers to a set of historical batches that share the same pathogen composition, inoculum preparation, seedling age, substrate treatment, and environmental window as the current inoculated batch, and whose group verification treatment results are valid. To ensure stable calibration results, the number of batches in the historical valid batch reference set should preferably be no less than 5, with an adjustable range of 5 to 20 batches. If the number of batches is less than the minimum, no new effective window lower limit, effective window upper limit, and allowable upper limit will be generated, and the verification caliber version that has been fixed in the previous round and has the closest conditions will be used. The effective window lower limit and effective window upper limit can be adjusted based on the quantile interval of the control response value in the historical valid batch reference set, preferably using the 10th percentile and 90th percentile values, or a fixed window that has been verified and adjusted by historical batches can be used; the allowable upper limit can be adjusted based on the upper quantile of the control offset value in the historical valid batch reference set, preferably using the 75th percentile or 90th percentile value. To avoid frequent fluctuations in the threshold within the same breeding cycle, the threshold is kept frozen when the number of newly added batches does not reach the preset number of batches. The preset number of batches is the implementation parameter, preferably 5 batches, with an adjustable range of 5 to 15 batches. After the preset number of batches is reached, the threshold is updated by sliding window, with the sliding window length preferably being 10 to 30 batches.

[0108] To ensure the feasibility of the calibration criteria, a field validation calibrator version number is added. This number is only used to bind the disease severity scoring criteria, root symptom scoring criteria, intra-batch composite weight, offset composite weight, effective window lower limit, effective window upper limit, and allowable upper limit used in the same set of validations, and is not included in the calculation. The historical effective batch reference set used for calibration should have the same pathogen composition, the same inoculum preparation, the same seedling age, the same substrate treatment, the same environmental window, and the same validation calibrator version number as the current batch. When the historical effective batches meeting the above conditions are less than the preset minimum sample size, no new effective window lower limit, effective window upper limit, and allowable upper limit are generated, and the validation calibrator version that has been fixed in the previous round and has the closest conditions is used. When there is no version that can be used, the current batch does not output a pass result, but is directly written to the set to be reviewed, and a batch offset recalibration identifier is attached. During calibration, historically valid batches that are completely identical to the current batch in terms of pathogen composition, inoculum preparation, seedling age, substrate treatment, and environmental window are prioritized as the reference set, and the threshold is kept unchanged within the same breeding cycle. When the cumulative number of newly added batches under the same conditions reaches the preset number, the threshold is re-estimated according to the sliding window. If continuous abnormal temperature and humidity fluctuations, concentrated mechanical damage to control plants, or concentrated non-pathogenic wilting occur, the threshold update is suspended, and the stricter threshold caliber of the previous round is used to avoid misjudging environmental disturbances as differences in resistance.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0110] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are meant to be within the scope of this application and form different embodiments. For example, all the embodiments above can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of this application and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. A method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot, characterized in that, include: According to the inoculation batches with records of pathogen composition, inoculum preparation, seedling age, substrate treatment and environmental window, multiple candidate lines of Coptis chinensis were inoculated with pathogens, and individual plant identification records, pedigree records, resistance observation records and sample transfer records were obtained; The resistance observation records are grouped into comparable batch groups according to the consistency of the inoculation batches. The resistance observation records of the same batch of Coptis chinensis control lines are used to generate group verification results. Only for comparable batch groups whose group verification results are in an effective state, the same batch of Coptis chinensis control lines, disease grade records, survival status records, and root symptom records are combined to form an intra-group retention set. When the same Coptis chinensis candidate line corresponds to multiple comparable batch groups, each of the comparable batch groups is used as an independent comparison unit, and the corresponding intra-group retention results are retained respectively. The candidate strains of Coptis chinensis and their control strains within the group are configured in a reference microecological layer and a non-reference microecological layer for stratified confirmation. When any configured microecological layer lacks a confirmation record, it is written into the rescreening queue. In the absence of any missing confirmation records, when the resistance level is consistent, or the resistance level is within the allowable fluctuation range relative to the reference microecological layer and maintains the same directional level difference relative to the control strain of Coptis chinensis in the same layer, and the range of inter-layer level difference does not exceed the upper limit of the allowable range of inter-layer level difference, it is written into the superior strain candidate library. When the resistance level is inconsistent, or the resistance level is consistent but does not maintain the same directional level difference, it is written into the observation queue. The corresponding results are associated with the pedigree record and the sample transfer record to generate seed retention task record, rescreening task record and delayed confirmation task record, respectively.

2. The method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot according to claim 1, characterized in that, The resistance observation records include disease severity records, survival status records, and root symptom records collected at the same observation point; The intragroup retention set is determined by first comparing disease severity records, then comparing survival status records, and finally comparing root symptom records to form the intragroup resistance level.

3. The method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot according to claim 1, characterized in that, When forming the comparable batch group, resistance observation records of the same batch of Coptis chinensis control lines are also obtained, and group verification results are generated based on the disease grade records, survival status records and root symptom records. The group verification results include valid state, weak state, strong state, and invalid state. Among them, the valid state is that the control lines of the same batch of Coptis chinensis form a pre-calibrated effective control response window within the specified observation window and the control deviation does not exceed the upper limit of the allowable limit. The weak state is that the overall disease incidence of the control lines of the same batch of Coptis chinensis is insufficient. The strong state is that the overall disease incidence of the control lines of the same batch of Coptis chinensis is too fast, the symptoms are concentrated and aggravated or the deaths are concentrated. The invalid state is that the control lines of the same batch of Coptis chinensis are missing, their identities are unclear, the observation records are missing, the observation results are inconsistent with the preset scoring direction, or the observation window is interrupted.

4. The method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot according to claim 3, characterized in that, When the current batch is determined to be in a valid state, the corresponding Coptis chinensis candidate strain will enter the group for screening; When a batch is determined to be in a weak, strong, or ineffective state, the corresponding Coptis chinensis candidate strain is added to the set to be reviewed. The original inoculation batch identifier and original resistance observation record are maintained until the review is completed, and the batch is stopped from participating in the screening within the current group.

5. The method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot according to claim 1, characterized in that, When the same Coptis chinensis candidate line corresponds to multiple comparable batch groups, the independent intra-group retention results are retained for each comparable batch group. The resistance observation records of different comparable batch groups are not included in the same comparison process, and a re-screening mark is generated for the Coptis chinensis candidate line.

6. The method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot according to claim 1, characterized in that, The sample transfer record includes root disease grade sample record, fibrous root sample record, rhizome sample record, rhizosphere sample record and aboveground phenotypic sample record, which are bound to the same single plant identification record. Each sample record corresponds to the sampling time, sampling location and transfer node. Before writing the candidate strains of Coptis chinensis into the candidate pool of superior strains, the screening queue, or the observation queue, an identity consistency check is performed on each sample record.

7. The method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot according to claim 6, characterized in that, The identity consistency verification includes detecting whether there are sampling location conflicts, transfer node conflicts, or batch affiliation conflicts among the sample records corresponding to the same single plant identification record. In the event of any conflict, a traceability review shall be performed based on the individual plant identification record, the source batch identification, the sampling time, the sampling location, the transfer node, and the material association key generated by the individual plant identification record and the pedigree record. When the unique correspondence can be restored after the source tracing and verification, the corresponding sample record is corrected and rebound, and the candidate strain of Coptis chinensis is restored to participate in the current group screening or stratified confirmation. When a unique correspondence cannot be restored, the corresponding Coptis chinensis candidate strain is written into the isolation review queue.

8. The method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot according to claim 1, characterized in that, The reference micro-ecological layer corresponds to a set of test plots with the same soil source and without the introduction of beneficial bacteria treatment; the non-reference micro-ecological layer includes the disturbing micro-ecological layer and the risk micro-ecological layer. The interfering microecological layer corresponds to a set of test plots where beneficial bacteria have been introduced and the soil source is consistent with the reference microecological layer. The risk microecological layer corresponds to a set of test plots where the continuous cropping status or pathogen spectrum is different from the reference microecological layer.

9. The method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot according to claim 1, characterized in that, When generating the seed retention task record, rescreening task record, or delayed confirmation task record, the seed retention order and propagation order are generated for the Coptis chinensis candidate lines written into the superior line candidate library. The corresponding inoculation batch identifier and microecological layer record are copied for the Coptis chinensis candidate lines written into the rescreening queue. The current pedigree position of the Coptis chinensis candidate lines written into the observation queue is retained and the next round of stratified confirmation arrangement is generated.

10. The method for resistance identification and systematic breeding of superior Coptis chinensis strains resistant to root rot according to claim 1, characterized in that, The stratified verification uses the same lineage of reserve or propagated seedlings that have been bound to the seedling inoculation stage. The same lineage of reserve or propagated seedlings are associated with the corresponding Coptis chinensis candidate lineage through the single plant identification record and the pedigree record. When a missing plant or sample appears in the configured microecological layer, and the reserve or propagated seedlings of the same strain can be continued within the current observation window, the original microecological layer is used to continue to confirm and maintain the association of the same material; when it is impossible to continue or the continuation exceeds the current observation window, a rescreening mark is generated and written into the rescreening queue.

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