Microsatellite Markers Determine the Genetic Structure and Diversity of Landraces of Quinoa from Ayacucho, Peru

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors. your work submitted to me for review is very interesting and I really appreciate your extensive research on a very important species of a crop plant such as quinoa. You have conducted very interesting studies that certainly required a lot of work and precision. The manuscript of your work is very interesting for me as a reviewer. I only have a few questions and suggestions regarding your research and possibilities of improving the manuscript before publication.

1. In lines 44-56 you mention many characteristics of Quinoa, but it is worth mentioning that it is a self-pollinating plant. Therefore, local populations limited geographically have genetic distinctiveness, because there is no wide genetic spillover between subpopulations. This is important for your study, because you have just compared varieties from different geographical regions and their genetic distinctiveness among themselves.

2.Lines 73-83 in this paragraph it is worth mentioning the importance of diversity within Quinoa and its importance in the global aspect. For this purpose I recommend reading the scientific work of Stanschewski, C.S.; Rey, E.; Fiene, G.; Craine, E.B.; Wellman, G.; Melino, V.J.; S. R. Patiranage, D.; Johansen, K.; Schmöckel, S.M.; Bertero, D.; et al. Quinoa Phenotyping Methodologies: An International Consensus. Plants 2021, 10, 1759. https://doi.org/10.3390/plants10091759 and possibly using it as a source in the literature

 

Comments on the Quality of English Language

The English language is correct, I have no objections.

Author Response

Reviewer 1

• Dear Authors. your work submitted to me for review is very interesting and I really appreciate your extensive research on a very important species of a crop plant such as quinoa.You have conducted very interesting studies that certainly required a lot of work and precision.The manuscript of your work is very interesting for me as a reviewer.I only have a few questions and suggestions regarding your research and possibilities of improving the manuscript before publication.

Response: We appreciate your comments and suggestions. These are very helpful. Thank you.

• In lines 44-56 you mention many characteristics of Quinoa, but it is worth mentioning that it is a self-pollinating plant.Therefore, local populations limited geographically have genetic distinctiveness, because there is no wide genetic spillover between subpopulations.This is important for your study, because you have just compared varieties from different geographical regions and their genetic distinctiveness among themselves.

Response: Thank you for your comment. We added “which possesses high self-pollination rate”. We also consider this pollination type in the Discussion section.

• Lines 73-83 in this paragraph it is worth mentioning the importance of diversity within Quinoa and its importance in the global aspect.For this purpose I recommend reading the scientific work of Stanschewski, C.S.;Rey, E.;Fiene, G.;Craine, E.B.;Wellman, G.;Melino, V.J.; R. Patiranage, D.;Johansen, K.;Schmöckel, S.M.;Bertero, D.;et al.Quinoa Phenotyping Methodologies: An International Consensus.Plants 2021, 10, 1759. https://doi.org/10.3390/plants10091759 and possibly using it as a source in the literature.

Response: Thank you for your suggestion. This reference is very useful!

We added “Quinoa and its wild relatives are estimated to be conserved in 59 genebanks located in 30 countries [25]. This germplasm represents a large genetic variability, possessing genes that may confer resistance to biotic and abiotic stresses [26]. This germplasm represents a large genetic variability, possessing genes that may confer resistance to biotic and abiotic stresses [26].”, “This nutritious crop still needs more research. Recently, an international network of quinoa researchers and trial datasets was established, linking around 300 members from more than 75 countries [26]. In addition, Stanschewski et al. [26] proposed an open-access framework to facilitate the systematic genetic, physiological and agronomic evaluation of quinoa aiming for its adaptation and breeding”

Reviewer 2 Report

Comments and Suggestions for Authors

Review of the manuscript titled “Microsatellite markers determine the genetic structure and diversity of landraces of quinoa from Ayacucho, Peru”.

The study is devoted to an important subject quinoa genetic diversity at the origin of this crop. The study used relevant methodology, obtained significant results which are well presented and interpreted. The paper contributes to the knowledge on the subject and deserves publication. However, there are avenues for its improvement.

1.      The material was collected from the farmers fields which is a great advantage. However, the authors did not describe the selection criteria for the landraces collection. Was it a a genetic resources collection mission with a broader objective of collecting and characterization.  What happened to collected seeds. Why nine landraces with the same name were collected from the same village (entries 53-61) and how the sampling could affect the diversity. Was any morphological description of the landraces made at collection or any socio-economic data of the farmers recorded. This information would put a study in a broader context of genetic resources and remove questions about the representation of the collected landraces.

2.      There is a confusion in relating genetic diversity and geography. On one hand the authors state: “quinoa from Ayacucho is grouped into three clusters without a clear geographic component”. The next sentence authors give the clusters geographical names of south and north. There was no diversity between administrative provinces but some diversity between the south-1, south-2 and north. Perhaps, the authors can ignore administrative provinces by try to characterize the three clusters using environmental variables (altitude, rainfall, growing season duration), agronomic practices and may be social variable, farm and field size, family size, years after coming back, etc. These efforts if successful would provide better meaning to the diversity clusters observed in the study.

3.      Figures 2 and 3 reflect the same phenomena and, perhaps, only one is needed.

4.      The discussion needs to avoid repetition of the results and look at a broader perspective. Are there breeding programs on quinoa in Peru, is there Gene Bank where these valuable resources maintained, and study information can be used.

5.      The main challenge of the paper its very narrow focus on limited number of markers using over 100 landraces which is fine for the paper to be published. However, the subject deserves much broader view due to its unique crop and environment.  

Author Response

Reviewer 2

• Review of the manuscript titled “Microsatellite markers determine the genetic structure and diversity of landraces of quinoa from Ayacucho, Peru”. The study is devoted to an important subject quinoa genetic diversity at the origin of this crop. The study used relevant methodology, obtained significant results which are well presented and interpreted. The paper contributes to the knowledge on the subject and deserves publication. However, there are avenues for its improvement.

Response: We appreciate your comments and suggestions. These are very helpful. Thank you.

 

• The material was collected from the farmers’ fields which is a great advantage. However, the authors did not describe the selection criteria for the landraces collection. Was it a a genetic resources collection mission with a broader objective of collecting and characterization. What happened to collected seeds. Why nine landraces with the same name were collected from the same village (entries 53-61) and how the sampling could affect the diversity. Was any morphological description of the landraces made at collection or any socio-economic data of the farmers recorded. This information would put a study in a broader context of genetic resources and remove questions about the representation of the collected landraces.

Response: Thank you for your comments. We added “Local quinoa varieties were randomly collected across different provinces, without selection preference.”. In the discussion section we also added “This UNSCH research initiative aimed to assess quinoa’s genetic diversity in Ayacucho after decades of social unrest in the 1980s and 1990s and a government program focused on commercial export varieties. The goal was also to establish a biotechnological platform for genetic improvement of the crop at UNSCH.”. Seeds are preserved at UNSCH. We added “The seeds are preserved at the Seed Germplasm Bank University”.

At the bottom of Table S1, we added “In the Andes, farmers name their cultivars as " quinoa" and in some cases the name is based on morphology and color in the Quechua language (e.g., black grains are called "Yana", red grains "'Puka", and yellow grains "Qello"). White grains are referred to as "Yuraq". If grains possess additional distinction, they are named for example "Yuraq choclito", which refers to its white color and a panicle resembling a maize cob. If no specific characteristic stands out, it's simply called "quinoa". The original farmer-given name is preserved during collection. Random sampling increases the chances of capturing greater genetic diversity, providing a representative sample of the population’s variability.

On the other hand, agromorphological characterization was performed using Bioversity descriptors on 85 local accessions under the conditions of Huamanga at 2750 masl, as reported by Alfaro Mendivil (2013) (undergraduate´s thesis). The agromorphological characterization of the remaining 20 accessions is projected to be completed. No socioeconomic data of the farmers were recorded.

 

• There is a confusion in relating genetic diversity and geography. On one hand the authors state: “quinoa from Ayacucho is grouped into three clusters without a clear geographic component”. The next sentence authors give the clusters geographical names of south and north. There was no diversity between administrative provinces but some diversity between the south-1, south-2 and north. Perhaps, the authors can ignore administrative provinces by try to characterize the three clusters using environmental variables (altitude, rainfall, growing season duration), agronomic practices and may be social variable, farm and field size, family size, years after coming back, etc. These efforts if successful would provide better meaning to the diversity clusters observed in the study.

Response: Thank you for your question regarding the characterization of the three groups, which allows us to point out that Ayacucho has a significant geographical divide between the northern and southern regions, due to its location in the Peruvian Andes and the lack of connecting transportation routes (See line 359). This was also pointed out by co-author Menéndez in his work (see Menéndez, 2016). We added “In southern Ayacucho, there is a deep-rooted cultural and political division between the “Rucanas” culture, now the Province of “Lucanas” (South-2), and the “Paucar del Sara Sara” basin (South-1), located further south, which may explain the grouping into South-1 and South-2. Furthermore, during the decades of social unrest, migration movements from northern and southern Ayacucho (South-2) were towards the Peruvian coast (mainly Lima and Ica), while those from South-1 migrated to Arequipa, deepening the separation between the three groups.

 

• Figures 2 and 3 reflect the same phenomena and, perhaps, only one is needed.

Response: With respect, we prefer to maintain both figures as they reflect different information regarding the three groups.

• The discussion needs to avoid repetition of the results and look at a broader perspective. Are there breeding programs on quinoa in Peru, is there Gene Bank where these valuable resources maintained, and study information can be used.

Response: Thank you for your questions. In Peru, there is a quinoa breeding program at the National Institute of Agrarian Research (INIA for its acronym in Spanish), which is limited to conducting traditional morphological and agronomic characterization, as well as pre-breeding activities. INIA possesses germplasm banks in different regions of the country, including Ayacucho. However, access to these banks is very limited. For this reason, we took the initiative to collect and initiate a germplasm bank for the university for research and breeding work.

• The main challenge of the paper its very narrow focus on limited number of markers using over 100 landraces which is fine for the paper to be published. However, the subject deserves much broader view due to its unique crop and environment.

Response: We appreciate your comments and suggestions. Our research group is applying for additional research grants so we can continue expanding our Germplasm Bank and also to conduct more genomics and phenomics work.

Reviewer 3 Report

Comments and Suggestions for Authors

The article titled "Microsatellite markers determine the genetic structure and diversity of landraces of quinoa from Ayacucho, Peru" provides a detailed analysis of the genetic diversity of quinoa landraces using microsatellite markers (SSR). However, it could be strengthened by addressing the deficiencies noted below. Improving the clarity, detail, and contextualization of the study will enhance its scientific rigor and impact.

1. Limited Geographic Scope:
• While the study is focused on Ayacucho, the authors acknowledge that further research is needed in other Peruvian regions. The study's narrow geographical focus limits its generalizability to broader quinoa populations in Peru and South America. Comparative studies with other Andean regions could have added value.
2. Low Expected Heterozygosity (HE):
• The study reports a low level of genetic diversity in terms of expected heterozygosity (HE), which is somewhat contradictory given the high allelic diversity reported. The low HE could be attributed to high inbreeding or genetic drift, but this discrepancy could have been explored further in the discussion. A deeper analysis of the cause of the low HE and its implications on quinoa diversity would strengthen the findings.
3. No Direct Comparisons with Other Genetic Studies in Peru:
• The study mentions previous work in other regions but lacks a detailed comparative analysis with studies conducted in regions such as Puno or Cusco. This would provide insights into how Ayacucho’s genetic diversity compares to other key quinoa-growing areas in Peru. Including these comparisons would enhance the article’s impact.
4. Admixture Discussion Could Be Expanded:
• The discussion around the limited admixture observed in the STRUCTURE analysis is brief. Given that quinoa is known for outcrossing, it would be beneficial to explore why such low admixture levels were found and what this indicates about the history of these populations.

The article provides a valuable contribution to understanding the genetic diversity of quinoa in Ayacucho, Peru. Its use of SSR markers and focus on the impact of genetic drift due to socio-political events makes it unique. However, expanding the scope to include other regions, a more detailed discussion on heterozygosity, discussing potential causes of low genetic diversity in more depth. Overall, it is a solid contribution to quinoa genetics but would benefit from broader geographical and methodological perspectives.

Author Response

Reviewer 3

• The article titled "Microsatellite markers determine the genetic structure and diversity of landraces of quinoa from Ayacucho, Peru"provides a detailed analysis of the genetic diversity of quinoa landraces using microsatellite markers (SSR). However, it could be strengthened by addressing the deficiencies noted below. Improving the clarity, detail, and contextualization of the study will enhance its scientific rigor and impact.

Response: We appreciate your comments and suggestions. These are very helpful. Thank you.

• Limited Geographic Scope:

While the study is focused on Ayacucho, the authors acknowledge that further research is needed in other Peruvian regions. The study's narrow geographical focus limits its generalizability to broader quinoa populations in Peru and South America. Comparative studies with other Andean regions could have added value.

Response: Thank you for your comment. Unfortunately, there are not many research papers published using Peruvian quinoa germplasm. Most studies were conducted at the thesis level (only available in Spanish) without publishing in refereed journals. Comparative studies with other Andean regions of Peru were analyzed and included in the discussion, specifically from Puno, Cusco, and Arequipa, as follows: Via Rada Fernandez in 2015 who evaluated germplasm from Puno and Cusco using SSR. We are comparing this with Cárdenas Córdova (2017), who evaluated germplasm from Puno, Allende Ciballero (2017), who evaluated germplasm from Cusco, and Christensen et al. (2007), who evaluated germplasm from Puno based on international germplasm. Additionally, from Arequipa, Romero (2019).

• Low Expected Heterozygosity (HE):

The study reports a low level of genetic diversity in terms of expected heterozygosity (HE), which is somewhat contradictory given the high allelic diversity reported. The low HE could be attributed to high inbreeding or genetic drift, but this discrepancy could have been explored further in the discussion. A deeper analysis of the cause of the low HE and its implications on quinoa diversity would strengthen the findings.

Response: Thank you for your comment. In the Discussion section, we added “Indeed, the low expected heterozygosity is attributed to the high rate of inbreeding and genetic drift, which may be explained by several decades of sociopolitical violence in the Ayacucho geographical department that forced entire farming populations to abandon their in-situ germplasm and migrate to other regions of the country. The returnees were mostly the children of migrants, who resumed quinoa cultivation with low heterozygosity, experiencing a bottleneck genetic drift. This, combined with the selection of commercial varieties supported by national programs for returning families, has generated the quinoa population structure presented in this article. Additionally, the reported allelic diversity is likely in homozygosity due to the self-fertilizing behavior of quinoa, reflected in the low expected heterozygosity.”

• No Direct Comparisons with Other Genetic Studies in Peru:

The study mentions previous work in other regions but lacks a detailed comparative analysis with studies conducted in regions such as Puno or Cusco. This would provide insights into how Ayacucho’s genetic diversity compares to other key quinoa-growing areas in Peru. Including these comparisons would enhance the article’s impact.

Response: Thank you for your comment. Unfortunately, there are not many research papers published using Peruvian quinoa germplasm. Most studies were conducted at the thesis level (only available in Spanish) without publishing in refereed journals. In addition, there are few published studies on quinoa diversity using SSR. Nevertheless, we are directly discussing results from related studies on quinoa diversity with SSR from Puno, Cusco, and Arequipa, such as Via Rada Fernandez (2015), who evaluated germplasm from Puno and Cusco with SSR. We compare this with Cárdenas Córdova (2017), who assessed germplasm from Puno, Allende Ciballero (2017), who evaluated germplasm from Cusco, and Christensen et al. (2007), who studied Puno germplasm based on international sources. Additionally, from Arequipa (Romero, 2019).

 

• Admixture Discussion Could Be Expanded:

The discussion around the limited admixture observed in the STRUCTURE analysis is brief. Given that quinoa is known for outcrossing, it would be beneficial to explore why such low admixture levels were found and what this indicates about the history of these populations.

The article provides a valuable contribution to understanding the genetic diversity of quinoa in Ayacucho, Peru. Its use of SSR markers and focus on the impact of genetic drift due to socio-political events makes it unique. However, expanding the scope to include other regions, a more detailed discussion on heterozygosity, discussing potential causes of low genetic diversity in more depth. Overall, it is a solid contribution to quinoa genetics but would benefit from broader geographical and methodological perspectives.

Response: Thank you for your comment. We added “Low levels of mixing could be explained as quinoa is primarily classified as self-fertilizing [4,72], with a small degree of outcrossing that varies by genotype. This self-fertilizing characteristic results in a population structure with little to no mixing, as demonstrated in this study.”. To broaden the discussion and information, we are including results from other regions/neighboring Andean countries, such as Chile (Fuentes et al. 2009), Anabalón Rodríguez (2018), Arenas Morales (2018), Bolivia (Del Castillo, 2007), Ecuador (Salazar et al. 2019), Colombia (Manjarres-Hernández 2022, and Argentina (Costa Tártara 2012).