MD Nizar Ahmad

Département Isabelle Boutron
email: isabelle.boutron@bch.ap-hop-paris.fr

PhD David Moher

MD Isabelle Pitrou

Carine Roy

MD Philippe Ravaud

External validity is neglected in reports of randomized trials The example of hip and knee osteoarthritis

Objective: To evaluate the reporting of data related to external validity (i.e., applicability of the results of a trial in routine practice) from randomized controlled trials (RCTs) assessing pharmacologic treatment and nonpharmacologic treatment for hip and knee osteoarthritis.

Methods: All RCTs assessing pharmacologic treatments (e.g., oral drugs, intra-articular injection, topical treatment) and nonpharmacologic treatments (e.g., surgery, rehabilitation, education, joint lavage, nonimplantable devices) for hip and knee osteoarthritis indexed between January 2002 and December 2006 were selected. A sample of 120 articles was randomly selected, 30 each of trials assessing pharmacologic treatments, surgery or technical interventions, rehabilitation and nonimplantable devices.

Results

The country where the trial took place was clearly reported in only 21% of reports (n = 25).

The setting was described in one-third of reports (n = 40) and the number of centres in 45% (n = 54). Details of centres (volume of care) were given in 20% of reports (n = 24). Reporting rates were lower for surgical trials for the country (3%), the setting (3%), the number of centres (13%), and details of centres (volume of care) (7%). The intervention was adequately described in all pharmacologic reports and more than 80% of reports of trials assessing rehabilitation. In reports of surgical intervention trials, the technical procedure was given in all reports, but the type of anaesthesia was reported in 13% (n = 4), pre-operative care in 7% (n = 2), and post-operative care in 50% (n = 15). In reports of device trials, the device was described in 93%, but the manufacturer was reported in only one-third of the reports. The method of recruitment was reported in 36% of reports (n = 43). Eligibility criteria were described in most reports (98%), but 22.9% of the exclusion criteria were poorly justified.

Conclusion: This study highlights the low reporting lack of data related to external validity in reports of RCTs assessing pharmacologic and nonpharmacologic treatments for hip and knee osteoarthritis.

BACKGROUND

Well-conducted randomized controlled trials (RCTs) are adopted as the gold standard for evaluating medical interventions [START_REF] Altman | The revised CONSORT statement for reporting randomized trials: explanation and elaboration[END_REF][START_REF] Schulz | Blinding in randomised trials: hiding who got what[END_REF][START_REF] Schulz | Allocation concealment in randomised trials: defending against deciphering[END_REF][START_REF] Schulz | Generation of allocation sequences in randomised trials: chance, not choice[END_REF]. For results to be clinically useful, RCTs must take into account the internal validity (i.e., the extent to which systematic errors or bias are avoided) and the external validity (sometimes called applicability -i.e., whether the results of a trial can be reasonably applied or generalized to a definable group of patients in a particular setting in routine practice) [START_REF] Rothwell | External validity of randomised controlled trials: "to whom do the results of this trial apply?[END_REF][START_REF] Glasgow | External validity: we need to do more[END_REF].

Historically, internal validity has been considered a priority for research. Several publications identified methods to avoid bias [START_REF] Moher | Does quality of reports of randomised trials affect estimates of intervention efficacy reported in metaanalyses?[END_REF][START_REF] Schulz | Empirical evidence of bias. Dimensions of methodological quality associated with estimates of treatment effects in controlled trials[END_REF]. The Consolidated Standards of Reporting Trials (CONSORT) Statements, endorsed by many major medical journals, improved on the reporting of data related to internal validity [START_REF] Altman | The revised CONSORT statement for reporting randomized trials: explanation and elaboration[END_REF][START_REF] Plint | Does the CONSORT checklist improve the quality of reports of randomised controlled trials? A systematic review[END_REF]. Tools [START_REF] Boutron | A checklist to evaluate a report of a nonpharmacological trial (CLEAR NPT) was developed using consensus[END_REF][START_REF] Higgins | Assessing risk of bias in included studies[END_REF][START_REF] Jadad | Assessing the quality of reports of randomized clinical trials: is blinding necessary?[END_REF][START_REF] Verhagen | The Delphi list: a criteria list for quality assessment of randomized clinical trials for conducting systematic reviews developed by Delphi consensus[END_REF] have been developed mainly to evaluate internal validity in reports of trial results included in systematic reviews [START_REF] Moher | Improving the quality of reports of meta-analyses of randomised controlled trials: the QUOROM statement. Quality of Reporting of Meta-analyses[END_REF].

Funders and journals have tended to be more concerned with the scientific rigor of interventions studied than with the applicability of the results. Consequently, external validity has been frequently neglected [START_REF] Glasgow | External validity: we need to do more[END_REF][START_REF] Bath | Quality of full and final publications reporting acute stroke trials: a systematic review[END_REF][START_REF] Bhandari | The quality of reporting of randomized trials in the Journal of Bone and Joint Surgery from 1988 through 2000[END_REF][START_REF] Dzewaltowski | Behavior change intervention research in community settings: how generalizable are the results?[END_REF]. This neglect has probably contributed to the failure to translate research into clinical practice. Lack of external validity is frequently advocated as the reason why interventions found to be effective in clinical trials are underused in clinical practice [START_REF] Rothwell | External validity of randomised controlled trials: "to whom do the results of this trial apply?[END_REF]. However, assessing the external validity of a trial to turn research into action supposes that information is adequately reported in published articles. Further, as highlighted by the extension of the CONSORT Statements to nonpharmacologic treatment, assessing external validity is probably more difficult for trials assessing nonpharmacologic treatments (e.g., surgery, technical interventions, rehabilitation, psychotherapy, devices) than pharmacologic treatments (e.g., oral drugs) [START_REF] Boutron | Methods and Processes of the CONSORT Group: Example of an Extension for Trials Assessing Nonpharmacologic Treatments[END_REF][START_REF] Boutron | Extending the CONSORT Statement to randomized trials of nonpharmacologic treatment: explanation and elaboration[END_REF].

The aim of this study was to evaluate and compare the reporting of external validity from RCTs assessing pharmacologic and nonpharmacologic treatments for hip and knee osteoarthritis (OA). We chose these conditions because they are highly prevalent and can result in disability and reduced quality of life. Further, international guidelines require the use of a combination of pharmacologic and nonpharmacologic treatments for optimal management of patients with these conditions [START_REF] Zhang | EULAR evidence based recommendations for the management of hip osteoarthritis: report of a task force of the EULAR Standing Committee for International Clinical Studies Including Therapeutics (ESCISIT)[END_REF][START_REF] Jordan | EULAR Recommendations 2003: an evidence based approach to the management of knee osteoarthritis: Report of a Task Force of the Standing Committee for International Clinical Studies Including Therapeutic Trials (ESCISIT)[END_REF].

METHODS

Search strategy and selection of reports

We identified all English-language reports of RCTs indexed between January 2002 and December 2006 in PubMed using the search terms "Osteoarthritis Hip" OR "Osteoarthritis Knee," with a limitation to RCTs, in MEDLINE via PubMed and to articles published in English. A similar search strategy was used in a previous work on internal validity [START_REF] Boutron | Methodological differences in clinical trials evaluating nonpharmacological and pharmacological treatments of hip and knee osteoarthritis[END_REF].

Eligibility criteria and screening process

We collected the electronic records in an Endnote data file. One of us (AN) assessed each reports by screening the titles and abstracts to identify relevant studies. A second person (IB) checked for adequate selection of the abstracts. Articles were included if the study was identified as an RCT assessing pharmacologic or nonpharmacologic treatment for hip or knee OA in a parallel-group or cross-over design. We excluded reports of cluster RCTs, nonrandomised trials, observational studies (cohort and case-control studies), extended follow-up trials (i.e., extended follow-up of patients included in an RCT beyond the last outcome assessment), nontherapeutic trials (metrological studies, epidemiological studies), pathophysiological studies, letters, an ancillary study of an RCT such as a subgroup analysis, cost-effectiveness evaluations, systematic reviews and/or meta-analyses. We also excluded reports of trials assessing the organization of the health care system or interventions provided to care providers. We excluded reports with these designs because we wanted to have a relatively homogeneous sample. The selected abstracts were classified according to the category of treatment assessed: pharmacologic treatments, surgery or technical interventions (e.g., joint lavage), rehabilitation, or nonimplantable devices.

For each category of treatment, we used a computer-generated list to randomly select 30 articles and retrieved the full text articles. Articles not fulfilling the inclusion criteria were replaced by a random selection of articles in the corresponding category. We chose a total of 120 articles for practical reasons, mainly to provide enough articles describing each category of treatment, and randomly selected articles to avoid selection bias.

Data collection

To assess external validity, as well as internal validity of the selected reports, we reviewed the literature and generated a standardized data extraction form (available upon request); we used items related to external validity proposed by the CONSORT Statement for RCTs(1), the extension of the CONSORT Statement for nonpharmacologic trials [START_REF] Boutron | Methods and Processes of the CONSORT Group: Example of an Extension for Trials Assessing Nonpharmacologic Treatments[END_REF][START_REF] Boutron | Extending the CONSORT Statement to randomized trials of nonpharmacologic treatment: explanation and elaboration[END_REF] and Rothwell et al. [START_REF] Rothwell | External validity of randomised controlled trials: "to whom do the results of this trial apply?[END_REF] Before data extraction, as a calibration exercise, the standardized form was tested independently by two members of the team (AN, IB) on a separate set of 20 reports. A meeting followed in which the ratings were reviewed and any disagreements were resolved by consensus. One reviewer (AN) independently completed all the data extraction. A random sample of 20 articles was reviewed for quality assurance.

The data extraction form covered the following data: 1) Characteristics of the selected studies: year of publication, journal, medical area of the study (i.e., hip OA, knee OA, hip and knee OA), type of treatment (i.e., pharmacologic treatment, surgical intervention, rehabilitation or education, nonimplantable device), type of control intervention (i.e., active intervention, placebo, usual care), funding sources (i.e., public, private, both, no funding, not reported or unclear), study design (i.e., parallel-group, cross-over) and sample size.

Internal validity of the selected reports was assessed with use of the following specific criteria recommended by the Cochrane collaboration and by most quality tools assessing results of pharmacologic and nonpharmacologic trials [START_REF] Boutron | A checklist to evaluate a report of a nonpharmacological trial (CLEAR NPT) was developed using consensus[END_REF][START_REF] Jadad | Assessing the quality of reports of randomized clinical trials: is blinding necessary?[END_REF]: allocation sequence generation; allocation concealment; blinding of patients, care providers and outcome assessors; and intent-to-treat (ITT) analysis.

2) External validity

The reporting of the following data related to external validity was evaluated:

Recruitment: data on the method of recruitment (i.e., referral from a rheumatologist or general physician, self-selection of patients through advertisement) and duration of recruitment.

Patients: data on patients' eligibility criteria as defined in a previous work [START_REF] Van Spall | Eligibility criteria of randomized controlled trials published in high-impact general medical journals: a systematic sampling review[END_REF], inclusion criteria (i.e., criteria governing entry or recruitment of individuals into the trial and describing the medical conditions of interest) and exclusion criteria (all other criteria limiting the eligibility of individuals) [START_REF] Van Spall | Eligibility criteria of randomized controlled trials published in high-impact general medical journals: a systematic sampling review[END_REF]. The exclusion criteria were classified as strongly justified, potentially justified and poorly justified reasons for excluding individuals from an RCT according to the classification proposed by Van Spall et al. [START_REF] Van Spall | Eligibility criteria of randomized controlled trials published in high-impact general medical journals: a systematic sampling review[END_REF]; exclusion criteria were considered strongly justified if an individual or substitute decision-maker was unable to grant informed consent, the intervention or placebo would likely be harmful, the intervention would likely be ineffective, or the effect of the intervention would be difficult to interpret.

Data on the number of eligible patients, number of patients not meeting inclusion criteria and number of patients refusing to participate were collected. We also checked whether the article reported baseline characteristics of excluded patients, as well as essential data on baseline characteristics of randomized patients (i.e., age, sex, weight/body mass index, ethnicity, coexisting diseases or co-morbidities, duration of the disease, measure of function status, level of pain, description of radiographic evidence of damage, and use of nonsteroidal antiinflammatory drugs).

Centre and care provider: data on the number of centers/care providers, expertise of centers/care providers and details of the centers (name, sources, organization, and expertise).

The reporting of the number of patients recruited in each center or by each care provider was recorded.

Intervention (i.e., whether and how details on the interventions were reported): for pharmacologic treatments, the route of administration, dosage, duration, frequency of treatment, and patient compliance; for rehabilitation, the number, timing, duration and content of each session, mode of delivery, supervision or not, and patient compliance; for surgical interventions, the type of anaesthesia, pre-operative care, post-operative care, description of the technical procedure and surgeons' compliance with the planned procedure; and for nonimplantable devices, the reporting of the manufacturer, description of the devices and patient compliance.

Abstract and discussion sections: information related to external validity reported in the abstract (i.e., country where the trial took place, setting, number of centers, number of eligible patients, number of patients randomized, length of recruitment, length of follow-up, and data on care providers) and whether the external validity was discussed in the discussion section of the study as recommended by the CONSORT Statement [START_REF] Altman | The revised CONSORT statement for reporting randomized trials: explanation and elaboration[END_REF].

Global assessment of external validity: quantitative assessment of external validity reporting may offer complementary information. Although it is difficult to specify which aspect of external validity is the most important, we decided to focus on 3 important components that are probably indispensable to assess the external validity of a trial: 1) the participants, 2) the description of the experimental treatment, and 3) the context of care (centres, setting, care providers' expertise). For each component, we identified items that were considered essential to allow an adequate assessment of the external validity of a published trial. These items are described in the box. The quantitative assessment of external validity was evaluated by the percentage of the selected items that were adequately reported, for each component.

Statistical analysis

All data analyses were completed using SAS for Windows, Release 9.1 (SAS Institute, Cary, NC).

We used descriptive statistics for continuous variables: means, standard deviation (SD), median (lower quartile; upper quartile) and minimum and maximum values. Categorical variables are described with frequencies and percentages. The results were adjusted for the clustering effect by journals as recommended [START_REF] Hewitt | Adequacy and reporting of allocation concealment: review of recent trials published in four general medical journals[END_REF]. The reporting of data related to external validity according to category of treatment was compared by a linear mixed-effects model, with the percentage of items with external validity as the dependent variable, fixed effects for treatment category, and journal as a random effect.

RESULTS

Articles selected

We identified 388 citations from our electronic search, of which 123 were excluded. Among the 265 included reports, we randomly chose 120 reports, 30 for each category of treatment.

After obtaining and reviewing the full text, 11 articles were replaced. The flow of articles through the study is presented in appendix 1.

Characteristics of the selected studies

Characteristics of the included studies are reported in table 1. The 120 articles were indexed in 53 journals. Among them, 13 (11%) were published in a general medical journal with a high impact factor and 107 (89%) in a general medical journal with low impact factor or in a special medical journal. Most trials, 118 (98%), had a parallel-group design. Three-quarters of the reports assessed knee OA (n = 90). The source of funding was described as public in 45 articles (38%), completely or partially private in 25 (21%). A funding source was not reported in 50 reports (42%).

The median sample size (interquartile range) was 100 (60-216) and was twice as high for reports of pharmacologic trials as nonpharmacologic trials.

The control group was described as receiving active treatment in 63 reports (52%), a placebo intervention in 43 (36%) and usual care in 14 (12%). Pharmacologic treatments and nonimplantable devices were mainly compared to placebo or active treatments, whereas rehabilitation interventions were mainly compared to usual care or active treatments, and surgical procedures were compared to active treatment in most reports.

The generation of allocation sequences was adequate in 51% of the reports. The treatment allocation was adequately concealed in 41% of reports (n = 49). Blinding was reported and was adequate in 43% of reports for patients, 32% for care providers and 59% for outcome assessors. An ITT analysis was described in only one-third of the reports.

External validity

The results for assessing external validity are reported in tables 2 and 3 and figure 1.

Recruitment (table 2)

The method of recruitment was described in about one-third of the reports; when described, this method relied on referral in 67% of reports (n = 29/43) and self-selection in 33% (n =14/43). The duration of recruitment was described in 47% of reports (n = 56); reporting was better in articles about rehabilitation. The median (interquartile range [IQR]) duration of recruitment for 10 patients per month described was 0.4 [0.2-0.8] months for pharmacologic trials; 0.8 [0.3-1.9] for device trials; 1.2 [0.9-2.7] for rehabilitation trials; and 2.5 [1.1-4.4] for surgical trials.

Participants (table 2)

Inclusion criteria were described in almost all reports (98%, n = 118) and exclusion criteria in 88% (n = 106). Exclusion criteria focused on age in 53% of reports (n = 64), medical comorbidities in 66% (n = 79), sex in 14% (n = 17), medication in 48% (n = 57), socioeconomic status in 2% (n=3), and patients participating in another trial in 5% (n=6).

Twenty-three percent of reports poorly justified exclusion criteria. These rates did not differ by category of treatment.

A flow diagram of participants through the trial was given in 40% of reports (n = 48).

Data related to the number of eligible participants, number of participants not meeting inclusion criteria and those refusing participation were reported in less than half of the reports, but reporting was better for rehabilitation trials. When given, the mean rate of participants not meeting inclusion criteria and refusing to participate was 22.5 (30%) and 19.2 (16%), respectively.

Baseline data of excluded participants were given in only one report. The baseline clinical characteristics of randomized participants were described in 91% of reports (n = 109); characteristics concerned age and sex in more than 80% of reports, weight or body mass index in 62%, and severity of disease (i.e., duration of the disease, pain, function, radiographic evidence of damage) in less than half. Patients' comorbidities were provided in only 12% of reports.

Interventions (table 3)

The treatments were described according to the CONSORT recommendations in all reports of pharmacologic trials and in most reports of rehabilitation trials but were missing in reports of devices and surgery trials. In the reports of medical device trials, a description of the device was given in 93% of reports (n = 28), but the manufacturer was stated in only 30% (n = 9). In the reports of surgical intervention trials, the technical procedure was given in all reports, but the type of anaesthesia was reported in only 13% (n = 4), pre-operative care in 7% (n = 2), and post-operative care in 50% (n = 15). Control treatment was described in most reports (98%, n = 117). Descriptions of co-interventions were lacking in 23% of reports (n = 28), mainly reports of pharmacologic trials.

Centres and care providers (table 2)

The setting was described in 33% of reports (n = 40) and the number of centres in 45% (n = 54). The country where the trial took place was clearly reported in only 21% of reports (n = 25). Details of centres were given in 20% of reports (n = 24). Other details such as centre sources, organization and expertise were never reported. The number of participants recruited in each centre was never reported. Details on care providers were given in 29% of reports (n =

35).

Abstract and discussion sections Information related to external validity was provided in the abstract of reports as follows: 4% of articles (n = 5) described the country where the trial took place, 15% (n = 18) the setting, 12% (n = 14) the number of centres, 2% (n = 2) the number of eligible patients, 92% (n = 110) the number of patients randomized, 5% (n = 6) the length of recruitment, 82% (n = 98) the length of follow-up, and 2% (n = 2) data on care providers. External validity was discussed in the discussion section of 11 articles (9%).

Global assessment of external validity (figure 1) Figure 1 highlights the reporting of each component of external validity by category of treatment. Reporting of essential baseline characteristics items was lower in reports of surgical trials, with a median [IQR] of 30% [START_REF] Khuri | Relation of surgical volume to outcome in eight common operations: results from the VA National Surgical Quality Improvement Program[END_REF][START_REF] Lavernia | Relationship of surgical volume to short-term mortality, morbidity, and hospital charges in arthroplasty[END_REF][START_REF] Mcgrath | Relation between operator and hospital volume and outcomes following percutaneous coronary interventions in the era of the coronary stent[END_REF][START_REF] Urbach | Does it matter what a hospital is "high volume" for? Specificity of hospital volume-outcome associations for surgical procedures: analysis of administrative data[END_REF][START_REF]Endarterectomy for asymptomatic carotid artery stenosis. Executive Committee for the Asymptomatic Carotid Atherosclerosis Study[END_REF][START_REF] Bond | Routine or selective carotid artery shunting for carotid endarterectomy (and different methods of monitoring in selective shunting)[END_REF][START_REF] Steg | External validity of clinical trials in acute myocardial infarction[END_REF][START_REF] Fortin | Randomized controlled trials: do they have external validity for patients with multiple comorbidities?[END_REF][START_REF] Coca | Underrepresentation of renal disease in randomized controlled trials of cardiovascular disease[END_REF][START_REF] Petersen | To whom do the results of this trial apply?" External validity of a randomized controlled trial involving 130 patients scheduled for primary total hip replacement[END_REF][START_REF] Gross | Reporting the recruitment process in clinical trials: who are these patients and how did they get there?[END_REF] of the essential items reported, than for those of trials of pharmacologic treatments, nonimplantable devices and rehabilitation, with a median [IQR] of 50% , 50% and 45% , respectively, of the essential items reported (p= 0.006).

The reporting of the intervention was better in reports of trials of pharmacologic treatments and rehabilitation (median [IQR] 80% [80-100] and 86% [71-100], respectively) than for those of trials of nonimplantable devices and surgery (median [IQR] 33% and 40% ), respectively; p <0.001).

The items dedicated to the context of the trial were poorly reported for trials of all treatments, especially pharmacologic treatments and surgery (median [IQR] 12% [START_REF] Jadad | Assessing the quality of reports of randomized clinical trials: is blinding necessary?[END_REF][START_REF] Verhagen | The Delphi list: a criteria list for quality assessment of randomized clinical trials for conducting systematic reviews developed by Delphi consensus[END_REF][START_REF] Moher | Improving the quality of reports of meta-analyses of randomised controlled trials: the QUOROM statement. Quality of Reporting of Meta-analyses[END_REF][START_REF] Bath | Quality of full and final publications reporting acute stroke trials: a systematic review[END_REF][START_REF] Bhandari | The quality of reporting of randomized trials in the Journal of Bone and Joint Surgery from 1988 through 2000[END_REF][START_REF] Dzewaltowski | Behavior change intervention research in community settings: how generalizable are the results?[END_REF][START_REF] Boutron | Methods and Processes of the CONSORT Group: Example of an Extension for Trials Assessing Nonpharmacologic Treatments[END_REF][START_REF] Boutron | Extending the CONSORT Statement to randomized trials of nonpharmacologic treatment: explanation and elaboration[END_REF][START_REF] Zhang | EULAR evidence based recommendations for the management of hip osteoarthritis: report of a task force of the EULAR Standing Committee for International Clinical Studies Including Therapeutics (ESCISIT)[END_REF][START_REF] Jordan | EULAR Recommendations 2003: an evidence based approach to the management of knee osteoarthritis: Report of a Task Force of the Standing Committee for International Clinical Studies Including Therapeutic Trials (ESCISIT)[END_REF][START_REF] Boutron | Methodological differences in clinical trials evaluating nonpharmacological and pharmacological treatments of hip and knee osteoarthritis[END_REF][START_REF] Van Spall | Eligibility criteria of randomized controlled trials published in high-impact general medical journals: a systematic sampling review[END_REF][START_REF] Hewitt | Adequacy and reporting of allocation concealment: review of recent trials published in four general medical journals[END_REF][START_REF] Bonell | Assessment of generalisability in trials of health interventions: suggested framework and systematic review[END_REF] and 25% [START_REF] Jadad | Assessing the quality of reports of randomized clinical trials: is blinding necessary?[END_REF][START_REF] Verhagen | The Delphi list: a criteria list for quality assessment of randomized clinical trials for conducting systematic reviews developed by Delphi consensus[END_REF][START_REF] Moher | Improving the quality of reports of meta-analyses of randomised controlled trials: the QUOROM statement. Quality of Reporting of Meta-analyses[END_REF][START_REF] Bath | Quality of full and final publications reporting acute stroke trials: a systematic review[END_REF][START_REF] Bhandari | The quality of reporting of randomized trials in the Journal of Bone and Joint Surgery from 1988 through 2000[END_REF][START_REF] Dzewaltowski | Behavior change intervention research in community settings: how generalizable are the results?[END_REF][START_REF] Boutron | Methods and Processes of the CONSORT Group: Example of an Extension for Trials Assessing Nonpharmacologic Treatments[END_REF][START_REF] Boutron | Extending the CONSORT Statement to randomized trials of nonpharmacologic treatment: explanation and elaboration[END_REF][START_REF] Zhang | EULAR evidence based recommendations for the management of hip osteoarthritis: report of a task force of the EULAR Standing Committee for International Clinical Studies Including Therapeutics (ESCISIT)[END_REF][START_REF] Jordan | EULAR Recommendations 2003: an evidence based approach to the management of knee osteoarthritis: Report of a Task Force of the Standing Committee for International Clinical Studies Including Therapeutic Trials (ESCISIT)[END_REF][START_REF] Boutron | Methodological differences in clinical trials evaluating nonpharmacological and pharmacological treatments of hip and knee osteoarthritis[END_REF][START_REF] Van Spall | Eligibility criteria of randomized controlled trials published in high-impact general medical journals: a systematic sampling review[END_REF][START_REF] Hewitt | Adequacy and reporting of allocation concealment: review of recent trials published in four general medical journals[END_REF][START_REF] Bonell | Assessment of generalisability in trials of health interventions: suggested framework and systematic review[END_REF]), respectively; p = 0.016).

DISCUSSION

This study assessed the reporting of external validity in a sample of 120 RCTs assessing pharmacologic and nonpharmacologic treatments for hip or knee OA during a 5-year period.

Our results highlight the lack of data related to external validity in published reports of RCTs.

Methods for recruiting patients were described in one-third of the reports; 22.9% of the exclusion criteria were poorly justified; important baseline data of patients were lacking; and setting, centers and care providers were described in one-third and less of articles. Further, the reporting of external validity differed depending on the category of treatment. Reports of trials assessing rehabilitation provided more adequate data related to recruitment, participants, setting and centers, and intervention. On the contrary, reports of trials assessing surgical procedures lacked such data, even though the reporting of some items, such as the setting, the number of centers and center volume, is particularly important in this field. In reports of pharmacologic trials and trials assessing nonimplantable devices, the reporting was of varying quality. In reports of pharmacologic trials, the reporting of the method of recruitment and of data related to centers and care providers was poor, but the reporting of the intervention was good.

To our knowledge, this is the first study that systematically appraised the reporting of data related to external validity from trials assessing pharmacologic and nonpharmacologic treatments. Most recent efforts of researchers and editors to improve the reporting of results of RCTs, such as the CONSORT initiative, have mainly focused on internal validity [START_REF] Altman | The revised CONSORT statement for reporting randomized trials: explanation and elaboration[END_REF][START_REF] Plint | Does the CONSORT checklist improve the quality of reports of randomised controlled trials? A systematic review[END_REF]. Nevertheless, external validity is also essential and needs to be emphasized [START_REF] Bonell | Assessment of generalisability in trials of health interventions: suggested framework and systematic review[END_REF][START_REF] Glasgow | Behavior change intervention research in healthcare settings: a review of recent reports with emphasis on external validity[END_REF]. The results of RCTs and systematic reviews cannot be relevant to all patients and all settings.

Consequently, reporting the results of RCTs should allow clinicians to judge to whom and in which context these results could reasonably be applied.

The setting, care providers and centers have obvious implications for external validity [START_REF] Rothwell | External validity of randomised controlled trials: "to whom do the results of this trial apply?[END_REF][START_REF] Moore | Surgical results: a justification of the surgeon selection process for the ACAS trial. The ACAS Investigators[END_REF].

In fact, the applicability of results of trials performed in secondary or tertiary settings applied to primary settings is often a concern [START_REF] Rothwell | External validity of randomised controlled trials: "to whom do the results of this trial apply?[END_REF]. Further, differences between health care systems can affect the applicability of results, especially regarding organization of care or reimbursement for the cost of care [START_REF] Rothwell | External validity of randomised controlled trials: "to whom do the results of this trial apply?[END_REF]. These issues are crucial in trials assessing nonpharmacologic treatments such as surgery or technical interventions. In fact, hospital and care providers' volume and outcome are related [START_REF] Halm | Is volume related to outcome in health care? A systematic review and methodologic critique of the literature[END_REF][START_REF] Hodgson | Relation of hospital volume to colostomy rates and survival for patients with rectal cancer[END_REF][START_REF] Khuri | Relation of surgical volume to outcome in eight common operations: results from the VA National Surgical Quality Improvement Program[END_REF][START_REF] Lavernia | Relationship of surgical volume to short-term mortality, morbidity, and hospital charges in arthroplasty[END_REF][START_REF] Mcgrath | Relation between operator and hospital volume and outcomes following percutaneous coronary interventions in the era of the coronary stent[END_REF][START_REF] Urbach | Does it matter what a hospital is "high volume" for? Specificity of hospital volume-outcome associations for surgical procedures: analysis of administrative data[END_REF]. A surgical procedure might be found to be safe and effective in an RCT performed in high-volume centres by high-volume care providers, but applying these results to low-volume centres might result in very different results [START_REF] Moore | Surgical results: a justification of the surgeon selection process for the ACAS trial. The ACAS Investigators[END_REF][START_REF]Endarterectomy for asymptomatic carotid artery stenosis. Executive Committee for the Asymptomatic Carotid Atherosclerosis Study[END_REF][START_REF] Bond | Routine or selective carotid artery shunting for carotid endarterectomy (and different methods of monitoring in selective shunting)[END_REF]. Surprisingly the reporting of data on care providers and centers was far less than optimal in our study, especially for trials assessing surgical procedures.

The representativeness of patients included in a RCT is also a major issue for external validity. The inclusion and exclusion criteria are among the greatest challenges in achieving representativeness of participants. Highly selective eligibility criteria can considerably reduce the applicability of the trial results. Our results highlight the lack of reporting of exclusion criteria in 12% of the trial reports; 23% of reported exclusion criteria were poorly justified.

These results are consistent with those of a systematic review of RCTs published in highimpact-factor journals between 1994 and 2006 [START_REF] Van Spall | Eligibility criteria of randomized controlled trials published in high-impact general medical journals: a systematic sampling review[END_REF]. Exclusion criteria reported in our articles concerned mainly elderly patients, those with medical comorbidities or those treated with specific categories of treatments. The exclusion of these specific categories of participants is problematic because it limits the representativeness of the patients.

The representativeness of the participants is also problematic because those agreeing to participate in RCTs often differ from those who do not participate. [START_REF] Steg | External validity of clinical trials in acute myocardial infarction[END_REF][START_REF] Fortin | Randomized controlled trials: do they have external validity for patients with multiple comorbidities?[END_REF][START_REF] Coca | Underrepresentation of renal disease in randomized controlled trials of cardiovascular disease[END_REF][START_REF] Petersen | To whom do the results of this trial apply?" External validity of a randomized controlled trial involving 130 patients scheduled for primary total hip replacement[END_REF]. Consequently, the number of eligible nonrandomized patients, as well as the number of participants who were invited to participate but declined, is important to adequately appraise the external validity of a trial [START_REF] Boutron | Methodological differences in clinical trials evaluating nonpharmacological and pharmacological treatments of hip and knee osteoarthritis[END_REF]; however, these data were reported in only one-third and one-quarter, respectively, of our reports, which is consistent with previous results [START_REF] Gross | Reporting the recruitment process in clinical trials: who are these patients and how did they get there?[END_REF].

Reporting the baseline clinical characteristics of participants included in RCTs should allow clinicians and others to assess external validity by comparison with their patients. Although baseline characteristics were described in almost all of our reports, some important data were missing: weight or body mass index, while essential, was given in only 62% of the selected articles. The reporting of ethnicity, comorbidities and severity and activity of the disease (pain, function, radiographic evidence of damage), which also predict response to and influence the generalisability of treatment was also inadequately reported [START_REF] Ettinger | Long-term physical functioning in persons with knee osteoarthritis from NHANES. I: Effects of comorbid medical conditions[END_REF][START_REF] Imamura | Does comorbidity affect the outcome of surgery? Total hip replacement in the UK and Japan[END_REF][START_REF] Kadam | Clinical comorbidity was specific to disease pathology, psychologic distress, and somatic symptom amplification[END_REF][START_REF] Kadam | Clinical comorbidity in osteoarthritis:Association with physical function in older patients in familly practice[END_REF].

External validity could also be affected if trials have treatment protocols that differ from usual clinical practice or have too stringent limitations on the use of cointerventions.

Further, to be able to adequately apply the results of the trial in clinical practice, the treatments should be described in detail to allow for adequate reproducibility. Our results highlight the lack of descriptions of nontrial treatments in two-thirds of the reports of pharmacologic trials and that descriptions of all the components of nonpharmacologic trials were lacking, especially in reports of surgery [START_REF] Jacquier | The reporting of randomized clinical trials using a surgical intervention is in need of immediate improvement: a systematic review[END_REF].

Finally, despite a specific item of the CONSORT Statement dedicated to external validity, very few articles considered this issue in the discussion section.

Our study has several limitations. First, we focused on the reporting of the trial, not its conduct. Consequently, these results highlight the lack of adequate reporting of external validity criteria and do not provide information on the applicability of the results of the trial.

Second, the results related to the rate of poorly justified exclusion criteria might be underestimated. In fact, some researchers have highlighted the inadequate reporting of eligibility criteria when comparing the published article with the protocol [START_REF] Shapiro | Reporting the study populations of clinical trials. Clear transmission or static on the line?[END_REF]; among an average of 31 eligibility criteria, only 63% were described in the main trial reports. Third, we focused on RCTs assessing hip and knee OA, and these results should be confirmed in other medical areas. However, we chose this disease because it is frequent and involves a wide range of pharmacologic and nonpharmacologic treatments. Further, the authors had some expertise in rheumatology and orthopedics and could therefore adequately evaluate the context of the trials.

In conclusion, this study highlights the lack of consideration of external validity in published reports of RCTs. Much attention is paid to the internal validity of clinical trials; however, even results of well-designed clinical trials are of limited use to clinicians if they have poor external validity and are not applicable to the patients for whom the intervention is designed.

Recently the CONSORT group developed an extension of the CONSORT Statements for pragmatic trials. This extension increases the focus on data related to external validity. This initiative should help improve the consideration of external validity.

Figure

  Figure1: Median percentage (interquartile range) of items of components of external validity that were reported in selected reports of pharmaceutical and nonpharmaceutical treatments for hip and knee osteoarthritis

Table 2 :

 2 1: Median percentage (interquartile range) of items of components of external validity that were reported in selected reports of pharmaceutical and nonpharmaceutical treatments for hip and knee osteoarthritis Number (%) of selected reports of trials of pharmaceutical and nonpharmaceutical treatments for hip and knee osteoarthritis that described items related to external validity

	A) BASELINE ITEMS

IQR: interquartile range

Legend

Box: Components of external validity evaluated in selected reports of trials of pharmaceutical and nonpharmaceutical treatments for hip and knee osteoarthritis Compliance of patients 9 [START_REF] Khuri | Relation of surgical volume to outcome in eight common operations: results from the VA National Surgical Quality Improvement Program[END_REF]


Gilles B Ena
email: gilles.bena@ird.fr

Paola A Fory

Johanna Echeverri Rico

Gloria M Mosquera

VNTR Typing of the Bacterial Rice Pathogen Burkholderia glumae Reveals the Coexistence of Several Diverging Lineages in a Single Field in Colombia

Keywords: Burkholderia glumae, Colombia, population structure, rice, VNTR

Burkholderia glumae is responsible for the panicle blight disease of rice. This disease is present worldwide and can result in significant drop in yields. To estimate the genetic diversity of the bacterial strains present in a rice paddy field in Colombia, we sampled 109 strains from infected panicles. To detect fine genetic relationships among related haplotypes, and to overcome a very low nucleotide diversity detected in previous studies, we designed primers to amplify and sequence several highly variable minisatellite loci, or variable number tandem repeats (VNTRs), as well as part of the Toxoflavin toxA gene in all strains. Results show that the toxA nucleotide diversity defined four lineages and was similar to that detected in several fields in Japan; data suggest that B. glumae has spread from Asia to America without major loss of genetic diversity, and that five VNTR loci discriminated the strains within the field revealing single and multi-infections of the rice panicles with a wide distribution of the haplotypes among the different plots. Even though disease levels vary considerably from year to year, the bacterial genetic diversity is maintained within a field. We do not detect any geographical structuring within the field, nor any effect of the rice cultivar on the observed diversity. The consequences on the origin and evolution of the bacteria are discussed.

B. glumae could become in the future the "next major pathogen of rice" [START_REF] Ham | Burkholderia glumae: next major pathogen of rice?[END_REF].

Data on the epidemiology of this disease remain very fragmentary. The bacterium is considered by some authors to be seed-borne, wherein it could be located in the epidermis or the parenchyma [START_REF] Agarwal | Principles of Seed Pathology[END_REF]. However, while B. glumae has been detected in seeds [START_REF] Cottyn | Phenotypic and genetic diversity of rice seed-associated bacteria and their role in pathogenicity and biological control[END_REF][START_REF] Sayler | Genetic characterization and real-time PCR detection of Burkholderia glumae, a newly emerging bacterial pathogen of rice in the United States[END_REF], seed transmission and the significance of seed-borne initial inoculum for the development of bacterial blight has not been formerly assessed. The bacterium might also be present in the leaf sheaths, forming the source of inoculum for development in the panicles, from which the spread of the disease would be in close proximity to other plants, therefore producing an aggregated profile. The spatial distribution within a field would be a function of the most affected panicles, from which the spread of the disease would be by proximity [START_REF] Trung | Occurrence of rice grain rot disease in Vietnam[END_REF]. The available data do not go much further, with only microscopy results that monitored tagged bacteria in planta [START_REF] Li | Infection process of Burkholderia glumae before booting stage of rice[END_REF]. Moreover, detection of the bacterium in the sheath, leaf blade, boot, or spikelet remains challenging, with drastic variations of population size from sheath to leaf [START_REF] Tsushima | Study on control and epidemiology of bacterial grain rot of rice[END_REF], leading to apparent "disappearance" and "reemergence" between consecutive stages of development [START_REF] Echeverri-Rico | Understanding the complexity of disease-climate interactions for rice bacterial panicle blight under tropical conditions[END_REF]. Finally, this bacterium has been shown to interact with other pathogens such as fungi, which may also play a role in the epidemiology of the bacteria [START_REF] Jung | Cooperative interactions between seedborne bacterial and air-borne fungal pathogens on rice[END_REF].

The understanding of the origin and emergence of this plant bacteriosis also remains very incomplete. The disease and its causative agent were described and isolated for the first time in Asia [START_REF] Kurita | On the pathogenic bacterium of bacterial grain rot of rice[END_REF]. However, as of this writing, there is no evidence that Asia is actually the center of origin for the disease. Furthermore, at local scale, data on the fine genetic diversity of the bacteria is almost nonexistent. One study has looked at the genetic diversity in one country. [START_REF] Maeda | Phylogenetic study and multiplex PCR-based detection of Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli using gyrB and rpoD sequences[END_REF] sampled and genetically typed 69 strains of B. glumae isolated from rice at 25 different sites in Japan. Characterization based on the sequencing of three loci was, however, not very informative on the genetic structure of the diversity of this species at this geographic scale because of the very low level of nucleotide diversity detected. With 25 isolates sampled in Arkansas in 2003, [START_REF] Sayler | Genetic characterization and real-time PCR detection of Burkholderia glumae, a newly emerging bacterial pathogen of rice in the United States[END_REF] detected 22 unique but closely related DNA fingerprint profiles. Cluster analysis revealed two major groups and their genetic diversity was relatively low as revealed by 16S-23S rDNA internal transcribed spacer sequence analysis. Finally, little genomic data are available. Indeed, at the time this project was initiated, only two full genomes were available from GenBank (including the type strain LMG2196 T ; https://www.ncbi.nlm.nih.gov/genbank/; [START_REF] Lim | Complete genome sequence of Burkholderia glumae BGR1[END_REF], five others being available as draft genomes. However, these strains do not correspond to any logical sampling (in an epidemiological or evolutionary sense), but rather are specific strains from different laboratories. Two studies have compared genomes [START_REF] Fory | Comparative analysis of two emerging rice seed bacterial pathogens[END_REF][START_REF] Francis | Comparative genomic analysis of two Burkholderia glumae strains from different geographic origins reveals a high degree of plasticity in genome structure associated with genomic islands[END_REF]) and suggested a considerable amount of plasticity, largely based on accessory genes and genome islands, including multiple insertion/deletion (indel) events.

The goal in this study was to get the first image of the genetic diversity of B. glumae isolates sampled from a single rice field in Colombia, a country where the disease has been present for several years with high incidence rates but with a high variation between years. Considering the lack of variable genotyping markers available for this species, we developed several B. glumae variable-number-of-tandem-repeat (VNTR; [START_REF] Bakhtiari | Targeted genotyping of variable number tandem repeats with adVNTR[END_REF]) markers to obtain a first snapshot of within field diversity, and thus circumvent the limits of the multi-locus sequence analysis approach. We detected a large genetic diversity, with several independent genetic lineages, raising the possibility of either several introductions of the bacterium into Colombia or considering this country as an alternative diversification zone.

Materials and Methods

Sampling of isolates. We sampled panicles from two rice varieties growing in a single field at the Fedearroz station in Saldaña, Tolima, Colombia (3 54950.800N; 74 5996.760W). In late summer in 2017, a trial included eight different varieties that were sown for agronomic and resistance to pathogens. Each variety was grown in four replicates, each in a 50 m 2 plot (Fig. 1). For each of the two varieties known to be either susceptible (F2000) or moderately resistant (FNAL004-1) to panicle blight, we selected in each plot three panicles from three different plants at maturing stage with, whenever possible, two plants with clear symptoms of panicle blight and one plant with no or low level of symptoms. Each panicle was stored in an individual paper bag.

Back at the lab, we isolated strains from seeds. Briefly, from five to 10 seeds were put in a 2-ml Eppendorf tube (Hamburg, Germany) and ground with a small sterile pestle. One ml of sterile water was added, and tubes were left at room temperature for 2 h. Serial dilutions of maceration, from 1/10 to 1/1,000, were plated (100 ll) on King's B agar [START_REF] King | Two simple media for the demonstration of pyocyanin and fluorescin[END_REF]) and incubated at 28 C during 24 h. Each plate was checked under a stereoscope and several colonies were picked up according to their morphology that were similar to B. glumae (pictures available upon request to G. B ena). All colonies collected were first streaked on King's B plates to verify their purity, and, starting from a single isolated colony, all were grown in liquid Yeast Mannitol Broth. DNA for each sample was extracted with a classical proteinase K protocol. All strains were stored at -80 C in 20% glycerol.

Molecular characterization of isolates. Toxoflavin, the main phytotoxin produced by both B. glumae and B. gladioli, is one of the main enzymes involved in the pathogenicity of B. glumae [START_REF] Jeong | Toxoflavin produced by Burkholderia glumae causing rice grain rot is responsible for inducing bacterial wilt in many field crops[END_REF] and, as of this writing, has never been detected in other species. In a first step, we tested for a positive amplification for all DNA samples targeting the methyltransferase toxA gene, which is part of an operon of five genes that codes for the synthesis of the phytotoxin toxoflavin, with primers designed in this study (Table 1). These primers were used both for confirmation of the taxonomic status of the isolate (positive or negative result for PCR amplification) and detection of genetic diversity among our isolates (by sequencing). All toxA and subsequent VNTR PCR products were sent to GenoScreen (Lille, France) for purification and forward strand sequencing.

In a second step, we typed all strains of B. glumae determined according to their toxA sequences. As previous works had shown the low genetic diversity detected among genomes available in GenBank or among isolates sampled all around Japan [START_REF] Maeda | Phylogenetic study and multiplex PCR-based detection of Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli using gyrB and rpoD sequences[END_REF], we used highly variable VNTR loci to characterize each strain. We used the online bioinformatics tool Polloc-V (http://bioinfo-web.mpl.ird.fr/ xantho/utils) to screen for VNTR loci in five B. glumae genomes from the NCBI data bank (LMG2196 T , BGR1, AU3208, 3252-8, and 336gr-1). Polloc-V identifies groups of loci based on the similarity rate of their flanking regions. In a first analysis, we used the following selection criteria: full loci size from 30 to 1,000 bp; pattern size from 5 to 9 bp; number of repetitions set to six or above; stringency set to maximal values 2 (match) -3 (mismatch) -5 (indel); and minimum percentage of similarity between repeat units = 80%.

In the first row of analysis, only a few primers could be selected, either because they could not be detected in one or several genomes (from indels or draft status) or because of their lack of diversity. We then perform a second analysis with the same parameters but with the pattern size extended from 10 to 90 bp (i.e., corresponding to minisatellites rather than microsatellites). Similarly, we only kept sequences that were recovered from the five genomes and for which diversity could be detected. For all loci detected that fulfilled these constraints, we designed a set of primers in the flanking regions of the tandem repeat sequence using the web interface tool, Primer3Plus (https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi; [START_REF] Untergasser | Primer3Plus, an enhanced web interface to Primer3[END_REF]. All pairs of primers designed were tested in silico using the suite of command-line tools NCBI-BLAST+ (https:// blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_ TYPE=Download; [START_REF] Camacho | BLAST+: Architecture and applications[END_REF]) against B. glumae genomes to confirm their specificity for the genomic region containing the locus. Whenever possible, melting temperatures were set around 60 C (Table 1). Moreover, because the genome of B. glumae consists of two chromosomes and four plasmids [START_REF] Lim | Complete genome sequence of Burkholderia glumae BGR1[END_REF], we tried as much as possible to have loci on each of the two chromosomes.

All primer pairs designed have been tested for positive amplification with a subsample of 24 randomly selected isolates from our derived sample. To obtain the highest level of diversity, including both the number of repeats and single nucleotide polymorphisms (SNPs), we sequenced these loci for all individuals. We retained for further analysis only those primers for which we obtained amplification for at least 95% of the individuals and for which sequencing revealed variability, both in terms of number of repeats and in terms of SNPs, both within the repeats and in the flanking regions of the locus. All sequences obtained were verified and corrected if necessary using the software ChromasPro v.1.34 (Technelysium Pty Ltd, South Brisbane, Queensland, Australia). The different haplotypes of each locus were detected using the tool Mothur [START_REF] Schloss | Introducing Mothur: Open-source, platform-independent, communitysupported software for describing and comparing microbial communities[END_REF]. Each different haplotype sequence has been deposited in GenBank. We finally included in the final dataset other B. glumae genomes (full or draft) that were released after the typing of all our isolates.

Data analyses. toxA sequence diversity. As a first step, using the tool BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_ TYPE=BlastSearch), we recovered all the B. glumae toxA sequences of at least 473 bp available in GenBank and we reconstructed a Maximum Likelihood phylogenetic tree using the program MEGA6 (https://www.megasoftware.net/; [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis version 6.0[END_REF]). The best model of molecular evolution was chosen according to the "Akaike's information criterion" function implemented in MEGA6. The toxA sequences of the sister species B. gladioli were only included when we searched for traces of selection along the branch joining the two species (see next paragraph). None of the data extracted from GenBank suggests toxA transfer between the two species, which confirms the species determination based on the toxA sequence (data not shown). As a second step, to simplify the phylogeny obtained, we used the software package Mothur (https://mothur.org). We kept only one or two representatives of each haplotype detected, retaining isolates from different countries when their sequences were identical to reflect the geographical distribution of diversity.

To explore a possible selection that would have shaped and constrained the nucleotide diversity of toxA sequences, we estimated the number of synonymous and non-synonymous mutations in the dataset using the tool DnaSP 6 (http://www.ub.edu/dnasp/; [START_REF] Librado | DnaSP v5: A software for comprehensive analysis of DNA polymorphism data[END_REF]. We tested for selection along the gene by estimating the dN/dS between all pairs of sequences (i.e., the ratio of the number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site). We also tested for a significant departure from a neutral evolution of this ratio along the branch joining the B. glumae and the B. gladioli clades using the online tool datamonkey (https://www.datamonkey.org/; [START_REF] Weaver | Datamonkey 2.0: A modern web application for characterizing selective and other evolutionary processes[END_REF] with the "aBSREL" test function (https://www.datamonkey.org/absrel; [START_REF] Smith | Less is more: An adaptive branch-site random effects model for efficient detection of episodic diversifying selection[END_REF].

VNTR diversity and network reconstruction. The different alleles of each VNTR locus were detected on the basis of the total length (and thus the number of repetitions) but also considered point mutations (same length but different sequences). The discriminatory power of each locus was compared with the Hunter-Gaston discriminatory index. This index is based on the probability that two unrelated strains randomly sampled in our population will be placed into different typing groups.

To visualize the different clonal complexes and genetic relationships among haplotypes, we applied a two-step procedure. We considered separately the nucleotide diversity detected in the toxA sequences and in the flanking region of each VNTR locus and the variations in the number of repeats for each VNTR locus. To begin, we retrieved all the nucleotide diversity from toxA and flanking regions, for which the rate of point mutations must be considerably lower than the rate of mutations within the repeat region. We concatenated these sequences for each isolate. With this data matrix, a neighbor-net split graph was built, using the software SplitsTree4 (http://www.softsea.com/review/ SplitsTree4.html; [START_REF] Huson | Application of phylogenetic networks in evolutionary studies[END_REF]. This graph allowed us to distinguish between the different groups of sequence type (ST) but also potentially illustrate recombination effects. Then, for the different groups of haplotypes obtained on the graph, we applied a network reconstruction using the program PHYLOViZ v.2 (https://www. phyloviz.net/; [START_REF] Nascimento | PHYLOViZ 2.0: Providing scalable data integration and visualization for multiple phylogenetic inference methods[END_REF], with the algorithm of global optimal ("goeBURST" function) distances. It was not a straightforward task, as one highly polymorphic VNTR locus (BG02_2) revealed, after sequencing either differences in length involving several indel events or alleles with a different nucleotide sequence (involving punctual mutations within repeats) but the same total number of repeats (i.e., homoplasy). We thus reconstructed the network based on the haplotypes coding schemes rather than on the VNTR length of each loci, and considered in the network all single and double locus variants.

Structure of the genetic diversity in the field. We tested whether there was a significant effect of the variety or plot on the bacterial genetic diversity recovered from each plant. Initially, we simply mapped on the haplotype network the origin of the isolates, whether it was the variety or the plot. We then tested whether the distribution of the different haplotypes between varieties and between plots deviated from a random distribution, using the program Genepop (https://cran.rproject.org; Rousset 2008), with the "population differentiation" option and an unbiased estimate of the P value performed on a contingency table. Subsequently, to disentangle plot and variety effects, we tested for a significant differentiation "between variety" and "among plot within each variety" in an analysis of molecular variance function

Results

Identification of B. glumae isolates and toxA diversity. We sampled 24 different panicles from eight plots and two varieties. Three panicles showed no symptoms (plots 103, 202, and 407), six showed mild symptoms, and the last 15 showed severe symptoms. We isolated strains based on their morphology from each of the panicles. The number of strains isolated per panicle varied from four to 14, and the number per plot varied from 24 to 31, for a total of 207 strains that were stored in glycerol at -80 C. The toxA amplification was positive for 119 samples. We sequenced all 119 of these PCR products, and sequences obtained clearly clustered 10 isolates within a B. gladioli clade with all other sequences of this species obtained from GenBank. Although known to be pathogenic on rice, we did not include these strains in the study because they represented <9% of the entire data sets and were not associated to a variety nor a specific plot. We continued the analyses with the remaining 109 isolates (Supplementary Table S1). These strains were distributed over the eight plots, with a variation from seven (plot 304) to 22 strains (106) per plot. For two panicles (one symptomless panicle from plot 103 and one with mild symptoms from plot 304) no strains for which toxA could be amplified were obtained. Conversely, we obtained six strains from a panicle in plot 202 that showed no symptoms (Supplementary Table S1).

We retrieved 88 toxA sequences from GenBank, originating from Japan, China, Korea, Vietnam, the U.S.A., Puerto Rico, and Colombia, corresponding to the main areas where the disease has been reported so far. We aligned these sequences with ours, which is 473 bp over the 738 bp of the entire toxA gene. Along the 473 bp we only detected five SNPs, two of which were specific to our dataset, giving seven different haplotypes, four of which were detected in our study. The alignment of 23 complete toxA sequences, extracted from GenBank with our four haplotypes, revealed only eight different mutations, four being singletons (i.e., limited to a single sequence). The phylogenetic reconstruction resulted in seven different clades, three including strains isolated from different continents (Fig. 2). Of the eight mutations detected, three resulted in amino acid substitution, while the five SNPs detected in our data are all silent. We did not detect any evidence of a significant departure from a neutral selection using the aBSREL test (https://www.datamonkey.org/absrel; [START_REF] Smith | Less is more: An adaptive branch-site random effects model for efficient detection of episodic diversifying selection[END_REF] implemented with the online tool datamonkey, either when testing the overall B. glumae phylogeny, or the branch joining the two B. glumae and B. gladioli species, or the branch joining the two Burkholderia species with two homologous sequences from Pseudomonas species (data not shown).

VNTR loci selection. We designed 14 pairs of primers that framed different VNTR loci. The quality and efficiency of these primers were first tested on DNA of the type strain LMG2196 T . Among the 14 pairs of primers, one pair did not produce any amplicon and was discarded. The 13 others each resulted in a single band of the expected size. We then tested these primers on a subsample of 24 B. glumae isolates randomly chosen from our study. The rate of success ranged from 42 to 100% for the primers, with only three primer pairs giving 100% amplifications. We retained these last three loci and a fourth primer pair, which failed to amplify two samples. Locus BG09 is located on chromosome 1 whereas the three others (BG02, BGm03, and BG12) are on chromosome 2.

We amplified and sequenced the four loci for all our isolates. Characteristics of each VNTR locus are given in Table 2. The length of each repeat (8 to 12 bp) classify them at the frontier between minisatellites and microsatellites. The BG02 locus is composed of two VNTRs in a row, the first with two to five repetitions of a 12-bp pattern, the second just afterward with 27 to 51 repetitions of a 9-bp pattern. In all subsequent analyses, we treated them separately, respectively as BG02_1 and BG02_2. Loci BG12 and BGm03 are nearly perfect, with only one SNP detected in each; BG02_1 is perfect, whereas BG09 and BG02_2 are compound VNTR loci, with, respectively, four and seven different repeats. However, in each case, the different repeats only differ from each other by a single nucleotide difference. The 109 typed strains produced 19 multi-locus haplotypes (Supplementary Table S2). The number of alleles, when considering both number of repeats and SNPs, ranged from six to 16 alleles (Table 2). When only considering number of repeats, the number of alleles ranged from four to 14. The HDGI scores ranged from 0.645 to 0.924. The combination of the five loci (toxA + the four VNTRs) has a discriminatory power of 0.938.

Within plot and within panicles diversity. Each sampled plot contains from two (plots 103 and 303) to six different haplotypes (plot 106). There is no correlation between genetic distance among haplotypes and their co-occurrence within a plot (Fig. 3). We can also note the low intrapanicle diversity observed. Indeed, of the 21 panicles for which we isolated several strains of B. glumae, only six contained several haplotypes (Supplementary Table S2). Among these six panicles, only one case (plot 106 panicle 2) suggests intrapanicular diversification (i.e., haplotype ST11). ST11, composed of only one isolate, is closely related to ST10 (n = 7 isolates) as a single locus variant, and further differs by a unique repeat. It probably emerged as a result of a mutation of ST10. The other panicles contain very divergent haplotypes differing by at least three VNTR loci, belonging to different genetic groups (i.e., groups including single-and double-locus variants) according to the network.

The four loci display different mutation step models. Sequencing suggested that each loci did not evolve by following the same mutation step model. Within the small clonal complexes, each allele differs from another only by a single repeat for VNTR loci BG12, BG09, BGm03, and BG02_01. Conversely, for BG02_02 we detected differences between alleles along the branches of clonal complexes ranging from one to 12 repeats. ST3 and ST12 each differ (by three repeats) at locus BG02_2 (38 and 41, respectively), and the alignment of the sequences shows that this is the result of a single three-repeat indel event. Conversely, ST1 and ST2 also differ by three repeats (34 and 31, respectively), but the alignment shows three different mutation events, involving three different indel events (Fig. 3). We mapped onto the network the different number of mutation events between haplotypes for BG02_2.

Network reconstruction and linkage disequilibrium. We reconstructed network haplotypes in a two-step analysis using the programs SplitsTree4 and Phyloviz v.2 (Fig. 3). The concatenated data of VNTR flanking regions and toxA sequences resulted in a 1,629-bp matrix with 35 polymorphic sites, including three singletons. In a first step, using the Neighbor-Net method implemented in SplitsTree4, we reconstructed a split network with four clearly separated groups of isolates, showing little evidence of recombination among them. In a second step, for each group, we reconstructed the network considering different alleles of each haplotype rather than the length of each VNTR locus to avoid homoplasic pitfalls (Supplementary Table S3). The tree cut-off, which is the threshold of number of differences (i.e., different alleles at each locus) below which haplotypes are considered to belong to the same clonal complex, was set to three. When considering only VNTR loci that have 19 different haplotypes and 171 potential links between them, 21 display <3 different alleles. The average number of different alleles for the five VNTR loci between haplotypes is 3.78, reflecting the genetic divergence among haplotypes. The network resulted in five groups and two singletons. Three groups each clustered together two haplotypes that differ between them at a single locus. The two other groups are more divergent, with up to two different alleles among haplotypes.

Considering all isolates, we detected a highly significant linkage disequilibrium among all loci (P < 10 -5 for each locus pair). The analysis of molecular variance showed a lack of variety effect when the plot effect was removed in the model (P = 0.26), reflecting the lack of specificity or preference of the various bacterial haplotypes by one of the two varieties.

Discussion

B. glumae is a bacterium that induces the rice panicle blight disease present in Asia and America and can occasionally cause extremely high yield losses. Our study explores for the first time in detail the diversity and genetic structure of bacterial populations in a cultivated rice field. The first step was to isolate bacteria from rice panicles. As expected, there is a strong correlation between the severity of the disease and our ability to recover isolates, even if we managed to recover a few isolates from asymptomatic panicles, as done in a previous study from China [START_REF] Zhu | Isolation and identification of Burkholderia glumae from symptomless rice seeds[END_REF]. Bacteria invaded the panicles and the grain and increased in density during infection [START_REF] Pedraza | Seed-born Burkholderia glumae infects rice seedling and maintains bacterial population during vegetative and reproductive growth stage[END_REF], which clearly explains our success in recovering more easily isolates from diseased panicles. We were, however, unable to detect any correlation between the genotype of the isolates and the health status of the panicle, although our sampling was not strictly adapted to detect such a link. Pathogenicity tests should be carried out to detect the variation in symptoms induced by each bacterial genotype and to see if some of them are more aggressive than others.

We recovered four different toxA haplotypes, involving four SNPs among the 109 isolates. Quite surprisingly, the study from [START_REF] Maeda | Phylogenetic study and multiplex PCR-based detection of Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli using gyrB and rpoD sequences[END_REF] typed 67 strains of B. glumae samples recovered from all over Japan, and in examining the same portion of the sequence as we did, only three SNPs and four haplotypes were found. The diversity of the partial toxA gene found in a single field in Colombia is therefore similar, if not higher, than the diversity found throughout Japan. Moreover, by reconstructing the phylogeny (although again based on a limited number of mutations), it appears clear that there is no global geographical pattern of toxA diversity (Fig. 2). For instance, toxA type 3 is shared by isolates recovered from the U.S.A. (336gr1), Vietnam (NCPPB3923), Japan (LMG2196 and others), Korea (BGR1), and Colombia (this study). It is also the case for type 2 (Japan and Colombia) and type 5 (Japan, and a lung isolate taken from a hospital in the U.S.A.). This finding demonstrates that, so far, the toxoflavin diversity is widespread over the different regions from which B. glumae isolates have been recovered. Because the majority of the mutations detected are silent (i.e., no change in the amino acid), we may reject the hypothesis that they have arisen independently by convergence and selection (i.e., are homoplasic). We can then suggest that if the disease really originated in Asia, the bacterium has spread from Asia to America, either once or repeatedly, without significant genetic bottleneck. Conversely, we can also suggest that Asia may not be the center of origin of the disease, which could be America instead. This strong hypothesis, however, requires a much larger sampling, both in Asia and America, to be tested. Finally, it can be noted that the majority of sequences recovered belong to haplotype 3 (59 sequences out of 87 in total), and that this haplotype is the one with the widest geographical distribution. Whether this predominance is the result of greater competitiveness in its ability to infest the host or to infest a wider host spectrum, to better survive in the environment (which could have led to a recent expansion), or is simply random should be tested by artificial inoculations and large sampling in other parts of the world.

It is finally interesting to note that the overall diversity, even over the whole sequence of toxA, remains extremely low. This low nucleotide diversity could be the result of high purifying selection because of its role in pathogenicity. We tried to detect evidence of such selection, but the test we applied was not significant; perhaps this was from the very low number of mutations along the sequences, which could restrict the power of this test. Whether these mutations have an effect or not, however, should be studied in depth in the future and their impact in the infection process should be assessed.

At the beginning of this project, we faced one main pitfall to select primers: only two genomes (LMG2196 T and BGR1) were completed at the time of this analysis; the three others remained at the draft level (from 96 to 2,457 scaffolds). The fragmentations of genome sequences Fig. 3. Network of the 19 haplotypes, reconstructed using the software programs SplitsTree4 and Phyloviz v.2. Within each subgroup detected in the SplitsTree software analysis, the network of haplotype was reconstructed using the different haplotypes of all variable numbers of tandem repeat (VNTR) loci and considering the two VNTR loci detected for the BG02 locus independently. The tree cut-off was set up at 3 (that is, we connected only sequence type [ST], which differed by one or two alleles). The thick line connecting STs indicates two differences (i.e., two different VNTR alleles) among them, while the thin line indicates a single locus difference among the five. Each number within a circle is the ST's haplotype code. Circle size is proportional to the number of isolates, with the range of variation of size given in the legend, from one to 14 individuals per haplotype. Colors indicate plot of origin of each haplotype, following the code given in the key (right). The VNTR code along branches between haplotypes indicate, in parentheses, the number of insertion/deletions between the two STs for BG02_2. For the other VNTR loci, it indicates the locus that differ by one-repeat variation between the two haplotypes. The dotted lines frame isolates that share the same toxA alleles (numbered from T1 to T4).

are often positioned within the repeated sequences, limiting the possibility of searching for sequences conserved on either side of the VNTR loci to design the primers. We did succeed in designing primers for 14 different loci, but many failed to amplify all of our subsample of isolates. Such a low level of success with our primers was quite disappointing. The primers were not simply designed on the basis of five genomes available in GenBank; we also verified that they were present in three other genomes added after our initial analyses. These eight genomes had been sampled from 1967 (for type strain LMG2196) to 2017 (Bp9029), taken from two continents and seven different countries. We were therefore expecting them to be representative of the species diversity, which was clearly not the case. As suggested earlier [START_REF] Francis | Comparative genomic analysis of two Burkholderia glumae strains from different geographic origins reveals a high degree of plasticity in genome structure associated with genomic islands[END_REF]), one explanation for this failure could be that B. glumae has a high degree of plasticity in its genomic structure, with several genomic islands. These rearrangements and indels could explain some of the amplification failures.

In a first stage of development of these VNTR loci, we chose to sequence all those that were usable to detect different variants although of the same length. As expected, considering all SNPs along sequences, rather than only the number of repeats, brought more information. Sequencing makes it possible to reveal undetected variants by simple analysis of amplified fragment sizes and thus increase the power of discrimination of the loci. This is visualized by the increase of the HDGI index. Although not sufficient to reconstruct fine genetic relationships among isolates, especially at the field scale, our typing scheme appears useful and was adapted to retrieve a first and reliable image of the genetic diversity detected from a single rice field. However, the combination of the four loci based on repeats number resulted in 18 different multi-locus haplotypes, whereas the full typing including SNPs resulted in 19 different haplotypes. The gain of information when considering point mutations is therefore limited in our study, although the number of different haplotypes increases for each VNTR locus. This is partly from the redundancy of information between loci as well as the very low level of recombination between loci (see below). We can, however, note that this set of VNTR loci is sufficient to visualize a one-off diversification event-with the ST11 haplotype that seems to have emerged within a panicle from the ST10 haplotype, which is detected within another plot as well.

The locus BG02 is highly variable, with 16 alleles detected (based on sequencing). BG02 is atypical compared with others in several features. First, as mentioned before, it is composed of two VNTR loci in a row, separated by a stretch of 13 nucleotides. It is also the single locus among the four that fall within a gene. It is part of a collagen-like protein, characterized by a Gly-Xaa-Yaa repeating amino acid sequence. This gene is similar to that found in animal collagens and underlies their unique triple helical structure [START_REF] Yu | Bacterial collagen-like proteins that form triple-helical structures[END_REF]. Collagen-like proteins in the two human pathogenic species, Burkholderia pseudomallei and B. mallei, have already been described by [START_REF] Bachert | A unique set of the Burkholderia collagen-like proteins provides insight into pathogenesis, genome evolution and niche adaptation, and infection detection[END_REF]. It was suggested that these proteins participate in pathogenesis, based on their non-collagenous domains that are associated with the components of a type-3 secretion apparatus. A type-3 secretion system is also present in B. glumae. However, even if the less virulent T3SS-deficient B. glumae mutant suggested that the effectors of T3SS are required and must play a role in the pathogenicity, to our knowledge none of them has ever been reported so far in B. glumae [START_REF] Cui | Research status and prospect of Burkholderia glumae, the pathogen causing bacterial panicle blight[END_REF]. Whether this collagen-like protein is involved in pathogenicity, and the repeated sequence is under selection, remains to be explored.

By aligning sequences for this locus, we detected several examples of homoplasy between haplotypes (showing the same number of repeats, but in different sequences) but found that differences in size between alleles may have resulted from several indel events. For instance, the two haplotypes ST5 and ST9 both have 45 repeats for the BG02 locus but with three indel events (located at different positions within the repeats) between them; this shows that their identical length is homoplasic and emerged independently. Similarly, ST4 and ST6 share 40 repeats but with a highly diverging pattern of indels between them. Conversely, ST13 and ST14 both display 39 repeats but with a single punctual nucleotide difference between them, suggesting their close evolutionary relationship and that the single nucleotide mutation has appeared recently in one isolate. A high number of loci analyzed may counterbalance homoplasy artifacts. However, in our case it could be easier and more useful to sequence this highly polymorphic and informative locus to enlighten us as to the evolutionary relationship among isolates rather than develop and type several other loci.

Based on five loci (splitting BG02 in two VNTR loci), we recovered 19 different haplotypes that could be clustered in seven different genetic groups as shown in Figure 3. However, the average number of locus variants among haplotypes taken from different groups reach 4.05, while the within group divergence is only 1.22. This large difference illustrates the discontinuity of genetic divergence observed between the different strains according to whether they belong to the same group or to different groups, reflecting the coexistence of several distinct genetic lines in this field. These values, together with the network structure and the linkage disequilibrium estimated, suggest, for this field, a lack of, or a very low level of, recombination among isolates. As we lack data from other sites, it is not clear to what extent this might be from an admixture of several independent lineages newly introduced into the field that will gradually recombine and homogenize over time and generations, or whether B. glumae is a species with a very low ability to recombine. One point that could partly resolve the issue is the geographical distribution of haplotypes in the field. After mapping the origin of each haplotype on the network, we found that there is no correlation between genetic proximity and the plot of origin. A plot may contain several divergent haplotypes, just as some haplotypes have been recovered from different plots (Fig. 3). Bacteria may disperse rapidly in the field (which is certainly possible). Alternatively, seeds used for the trials may have been heavily contaminated at the time of sowing. Indeed, previous studies have shown that seeds may contain B. glumae isolates [START_REF] Cottyn | Phenotypic and genetic diversity of rice seed-associated bacteria and their role in pathogenicity and biological control[END_REF][START_REF] Sayler | Genetic characterization and real-time PCR detection of Burkholderia glumae, a newly emerging bacterial pathogen of rice in the United States[END_REF]). However, because there is no effect of the rice variety on the bacterial diversity recovered, we had to hypothesize that the two lots of seeds have been produced under the same bacterial population contamination pressure. Finally, this distribution on the different plots could suggest that these different haplotypes have been present for a long time, and therefore would mean that the degree of recombination among strains must remain very low.

These results suggest key elements in the presence of the bacterium in Colombia. Colombia is a region of major diversity and diversification of B. glumae as a rice pathogen, with the genetic diversity observed being the result of migration events from Asia (the region assumed to be the center of origin of the pathogen) and also possibly from other regions where the disease is present, such as northern America. Indeed, the presence of toxA haplotypes shared between Asia and Colombia, coupled with the high conservation of sequences of this gene, supports the hypothesis of recurrent migration events between the two continents. This is not surprising, based on the high exchange of rice germplasm for breeding purposes. At the same time, the high number of multi-loci haplotypes and VNTR alleles reflect the diversification process that occurs in Colombia.

If we set aside the hypothesis that the seed lots used were already heavily contaminated, the coexistence of several genetically highly divergent bacterial lineages within a field shows that these bacterial lineages are locally maintained and can reinfest a field year after year, either by wind or by irrigation water. Such a dynamic must play a great role in the epidemiology of the disease and its ability to maintain itself over years in a geographic area. It would be particularly relevant to study the extent to which such diversity may have a role in the dynamics of the disease and its adaptability to the different rice varieties that can be deployed in the field. We have not yet tested for differing virulence or aggressiveness in rice cultivars. However, it certainly might be the case, because previous studies have shown that different strains may have variable toxoflavin production, which affects their ability to colonize the plant, and their particular virulence [START_REF] Karki | Diversities in virulence, antifungal activity, pigmentation and DNA fingerprint among strains of Burkholderia glumae[END_REF]. It has been also reported that B. glumae can act as a biocontrol agent (some strains are producers of antibiotics) and that may help for survival in the seeds and other tissues [START_REF] Cottyn | Phenotypic and genetic diversity of rice seed-associated bacteria and their role in pathogenicity and biological control[END_REF]. Colombia experiences large year-to-year variations in disease incidence and severity. Our results suggest that this is not from annual variations in the diversity of strains present but more likely the result of environmental fluctuations; previous studies have shown that the disease is dependent on particular environmental conditions for its development.

Fig. 1 .

 1 Fig. 1. Map of the trial established during September 2017 in the Experimental Center las Lagunas -Fedearroz in Saldaña, Colombia. Each plot (50 m 2 each) is numbered from 1 to 8, with four replicates (10 to 40). The whole field was flooded. Rice genotypes shown in each plot are in italics. Plots in gray shading were sampled. The blue cross in a circle is where the global positioning system track was taken (3 54950.800N; 74 5996.760W).

Fig. 2 .

 2 Fig. 2. Maximum likelihood phylogeny of the toxoflavin toxA sequence. Tree is based on partial 473-bp sequences. The seven types detected are numbered from 1 to 7, while numbers in parentheses and italics indicate the number of sequences retrieved from GenBank that fall in each clade (thus excluding sequences from our study). Types 2, 3, and 5 clustered sequences are from different countries.

Table 1 .

 1 List of the primers used, all of which were designed in this study with the program Arlequin v.3.0 (http://cmpg.unibe.ch/ software/arlequin3/;[START_REF] Excoffier | Arlequin (version 3.0): an integrated software package for population genetics data analysis[END_REF]. The linkage disequilibrium among all pairs of loci was tested with the "log likelihood ratio" statistic implemented in the program Genepop.

	Locus	Primer's name	Primer's sequences (59 to 39)	T m a /elongation time	Location (chromosome/gene) b
	ToxA	ToxAF	TTTCGGGCGTGAAATCTATC	56 C	Chr2; toxoflavin methyltransferase
		ToxAR	GTCTGCAGGCAGTTGTTCTG	30 s
	BG09	BG09F	AAACGGTTCCGATTTTCCTC	57 C	Chr1; hypothetical protein
		BG09R	TGGTGGTCGACAACGACAG	1 min
	BGm03	BGm03F	ATCATGGACCGGTCTCTCC	57 C	Chr2; intergenic sequence
		BGm03R	TCCGCGTTCTAACTTCTACGA	1 min
	BG12	BG12F	CGGCCGATGTCCTTAAAC	57 C	Chr2; intergenic sequence
		BG12R	TAGTATTGACCGGCGTGAAA	1 min
	BG02	BG02F	GTCGGYATATCGTCGATGC	57 C	Chr2; lipoprotein, bacterial collagen
		BG02R	GTGAAAACCGTGCCGAGT	1 min 30 s

a Tm, melting temperature. b Location on chromosome and gene are given according to LMG2196 T genome sequence and annotation (GCA_000960995.1); all PCR cycling parameters start with an initial denaturation step (95 C for 5 min) followed by 30 three-step cycles (94 C/30 s, Tm/30 , 72 C/elongation time). performed

Table 2 .

 2 Genetic diversity of the five loci retained in the typing scheme of Burkholderia glumae a

			Number of alleles		Allelic range
		Number of	based only on	Number of SNPs b	variation	Type of	PCR product	HGDI b
	Locus	alleles	repeat numbers	in flanking regions c	(size of the repeat)	VNTR	size range (bp)	scores
	toxA BG09	4 9	-8	0 13	5-19 (9 bp)	-	-C b (4 types)	560 522-648	0.698 0.872
	BGm03	6	4	6	2-5 (10 bp)		P b (1)	404-445	0.745
	BG12	7	5	7	2-11 (8 bp)		P (1)	460-532	0.795
	BG02	16	14	4	2-5 (12 bp) and 27-51 (9 bp) d	P and C (7)	312-528	0.924

a The basic statistics were obtained from the Saldaña collection (n = 119). b SNP, single nucleotide polymorphism; VNTR, variable number of tandem repeats; HGDI, Hunter-Gaston discriminatory index; P, perfect (numbers of SNPs detected within VNTR); C, compound (number of types detected). c From 68 to 394 bp around the VNTR loci. d Locus BG02 includes two VNTR loci in a row.

Acknowledgments

We are very grateful to L. Gagnevin and two anonymous reviewers for their helpful comments on an earlier version of this manuscript and to Isabelle Rimbault for technical help in the lab work.

This work was supported by the Consultative Group on International Agricultural Research (CGIAR)'s Program on Rice Agri-food Systems.

*The e-Xtra logo stands for "electronic extra" and indicates there are three supplementary tables published online. The author(s) declare no conflict of interest.