Physiological and genetic characterization of heat stress effects in a common bean RIL population

Heat stress is a major abiotic stress factor reducing crop productivity and climate change models predict increasing temperatures in many production regions. Common bean (Phaseolus vulgaris L.) is an important crop for food security in the tropics and heat stress is expected to cause increasing yield losses. To study physiological responses and to characterize the genetics of heat stress tolerance, we evaluated the recombinant inbred line (RIL) population IJR (Indeterminate Jamaica Red) x AFR298 of the Andean gene pool. Heat stress (HS) conditions in the field affected many traits across the reproductive phase. High nighttime temperatures appeared to have larger effects than maximum daytime temperatures. Yield was reduced compared to non-stress conditions by 37% and 26% in 2016 and 2017 seasons, respectively. The image analysis tool HYRBEAN was developed to evaluate pollen viability (PolVia). A significant reduction of PolVia was observed in HS and higher viability was correlated with yield only under stress conditions. In susceptible lines the reproductive phase was extended and defects in the initiation of seed, seed fill and seed formation were identified reducing grain quality. Higher yields under HS were correlated with early flowering, high pollen viability and effective seed filling. Quantitative trait loci (QTL) analysis revealed a QTL for both pod harvest index and PolVia on chromosome Pv05, for which the more heat tolerant parent IJR contributed the positive allele. Also, on chromosome Pv08 a QTL from IJR improved PolVia and the yield component pods per plant. HS affected several traits during the whole reproductive development, from floral induction to grain quality traits, indicating a general heat perception affecting many reproductive processes. Identification of tolerant germplasm, indicator traits for heat tolerance and molecular tools will help to breed heat tolerant varieties to face future climate change effects.


Introduction
The introduction covers the main points of the study, however, the lack of zeal in the accuracy of the references cited and in the formatting of the file is evident. The subject of QTL mapping was presented in a confusing and poorly introduced way, missing the link between the paragraphs.
According to the journal style, citations must be between "[]" and not "()". Please review the entire file. L51 -The reference "(5)" does not seem to support the affirmation, so it is not necessary. L52 -Add "the" before "effects".
L52 -The phrase "However, climatic conditions are shifting due to effects of climate change" sounds strange.
L56 -The phrase "Current research suggests that the average global temperatures have increased by ~0.8°C since 1880" need to be referenced. L60 -Change "as" by "than".
L63 -The introduction paragraphs must not be separated with empty lines. Please review the entire file (L72, L77, L83, L94).
L65 -The phrase "High nighttime temperatures during the reproductive phase cause heat stress in common bean, 66 and to a lesser degree, high daytime temperatures" need to be referenced. L67 -Add "the" before "abortion". L70 -Add "the" before "night".
L73/76 -I did not find any results in the reference "(16)" that supports the paragraph "Common bean genotypes of the Andean gene pool are commonly grown at mid to midhigh altitudes (1400-2800 masl) or in cooler climates, whereas genotypes of the Mesoamerican gene pool adapt to low to mid altitude ranges (400 -2000 masl) with higher temperatures. For this reason, Andean beans are expected to be more sensitive to high temperatures (16)." L79 -Add references of studies carried out "under stress conditions" and "controlled environments", separately. L84/88 -The paragraph is very confused and poorly structured. The QTL mapping makes it possible to identify loci associated with the trait of interest, in order to provide information on markers linked to the QTL that can be used for SAM. However, it is necessary to clarify that SAM and mapping are two totally different approaches.
References are also missing.
L90 -Change the reference by the journal style.
L92/93 -There is no point in discussing MAS if the work did not aim at MAS. It is preferable to improve the discussion of the importance of identifying markers associated with QTLs, aiming at the use of SAM.

Materials and methods
Although a population with 107 genotypes is considered small for linkage mapping studies, it is necessary to consider the complexity of assessments for hightemperature stress. I have no experience with alpha lattice design and therefore I am not able to make considerations. However, in my opinion the NS2018 and HS2017 trials should not be considered for the study since both have no design and repetition. L122/128 -It is necessary to make it clearer that the variation of the maximum temperature mentioned, refers to the variations of the maximum temperatures of each day in relation to the total period of evaluation. The same for the minimum temperature variation.
L134 -What means "experimental plots were not replicated"? For HS2017 the 3 repetitions mentioned for HS2016 were not adopted? I did not understand.
L143 -Was the trial irrigated? If so, how many times a day? Likewise for all trials? L145 -Add "period" or "time" after "flowering". L160 -Change the phrase by "Finally, to determine the percent viability (number of grains stained with respect to the total), the pollen grains were count using the software HYRBEAN". L162 -Change "read" by "evaluated".
L166 -The caption must be more complete, adding the name of the species or "common bean", the name of the genotype used for the example and etc. L237 -Change "y" by "and".
L258/264 -The number of SNP filtered both for the polymorph of the parents and for the redundancy test can be presented in the results section.
L274 -The graph used is very good! It clearly shows that there was a contrast between both environments (HS and NS). 279 -Change "or" by "and". L280/281 -One more reason not to consider HS2017 data.
The manuscript needs a review of English and formatting for the style of the journal, so I do not comment further on these errors.
L282 -"Onset of flowering was noted about 5 days later in the HS trials than in NS conditions." Was the germination day of the plots recorded? Germination in the field depends intrinsically on soil moisture. Please provide more information about this if the authors want to discuss the difference in the number of days for flowering.
L319/320 -The affirmation is not valid. Although most correlations are significant (4 non-significant), the highest significant correlation was 0.5 and the lowest was 0.23 (mean 0.34). These values do not support that the data are of sufficient quality to contribute to analysis.
L376 -Missing data rate less than 0.5 is very high. Usually a filter is applied around 0.8 to 0.9. Especially for Beadchip technology, where the genotyping error rate is usually very low.
L385 -Poor caption, information on the number of population genotypes, number of markers, difference between bars representing QTL and so on is lacking.
L399/401 -This belongs to the discussion.

Discussion
The discussion, besides having several formatting errors, both in the text and in the references, has several statements that need citations. In addition, based on the complexity of the trials and the magnitude of the traits evaluated, many more things could be discussed and discussed.
L454/455 -The statement is not entirely true. It is worth mentioning that although the controlled environment does not reflect the real conditions, the evaluation in a controlled environment for a study whose main objective is "Physiological and genetic characterization" would be ideal. Field conditions being the best for selecting superior strains.
L466/467 -Due to the difference in conducting the trials, it is not possible to compare HS2016 with 2017 and conclude that nighttime temperatures have major importance in heat stress. L504/505 -Provide the reference.