Role of Genome Sequences of Major and Minor Millets in Strengthening Food and Nutritional Security for Future Generations

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Dear editor and dear colleagues,

most of the requested data have been reported in the revised version, and i believe that the manuscript is improved

I have no further comments

-

Author Response

Many thanks for your time to review the revised article and for approving it for publication.

Reviewer 2 Report (New Reviewer)

This article presents a comprehensive review on the role of genome sequences in major and minor millets, aiming to enhance food and nutritional security for future generations. It discusses the genetic resources of millets, including genome sequences, QTLs, functional gene, and their potential applications in crop improvement. This includes increasing resilience to environmental stresses, enhancing nutritional profiles, and boosting crop yields. The overall architecture of the manuscript is fundamentally sound, yet there are sections that, in my view, still necessitate refinement.

1. A significant issue with this article is that it overlooks several important advancements in millet research that have occurred in recent years. This includes pan-genome studies on foxtail millet and sorghum, as well as the Telomere-to-Telomere (T2T) genomes for these species.

2. Many sections of the article do not reflect the most recent developments, such as QTLs, GWAS studies.

3. In Section 9, the genes discussed are predominantly derived from gene family analyses and omics datasets. I contend that the utility of these genes as references is markedly limited, and thus, they should not be emphasized as a principal component of the manuscript. Actually, remarkable progress has been made in the molecular biology of foxtail millet and sorghum in recent years, progress that this manuscript fails to acknowledge or incorporate.

Author Response

General Comments

This article presents a comprehensive review on the role of genome sequences in major and minor millets, aiming to enhance food and nutritional security for future generations. It discusses the genetic resources of millets, including genome sequences, QTLs, functional gene, and their potential applications in crop improvement. This includes increasing resilience to environmental stresses, enhancing nutritional profiles, and boosting crop yields. The overall architecture of the manuscript is fundamentally sound, yet there are sections that, in my view, still necessitate refinement.

Ans. Thank you for your positive response to our manuscript. We would like to thank the reviewer for further valuable comments to improve the manuscript. We have addressed all the comments raised by the reviewer and improved the manuscript.

Minor Comments

Q1. A significant issue with this article is that it overlooks several important advancements in millet research that have occurred in recent years. This includes pan-genome studies on foxtail millet and sorghum, as well as the Telomere-to-Telomere (T2T) genomes for these species.

Ans. Thank you for providing such a valuable comment to improve the manuscript. As suggested by the reviewer, we added separate section to the revised manuscript that talk about "Pan-genomic and telomere-to-telomere genome resources of major and minor millets." These sections were based on the reports that were already out there. The details of the newly added contents are shown below.

“7. Pan-genomic and telomere-to-telomere genome resources of major and minor millets

The available genomic resources for millets have been developed from a single germplasm line/genotype of millets, which provides genetic information on a single gen-otype. Pan-genome studies will enable the simultaneous generation of genome resources for different cultivars, landraces, and wild ancestral species [55]. This will allow research-ers to search for novel genes and alleles that may have been inadvertently lost in domesticated crops during the historical process of crop breeding or extensive plant breeding. Pan-genomic resources are currently available for two major (sorghum and pearl millet) and two minor (foxtail millet and proso millet) millets. Pan-genome analysis of 13 genetically diverse genotypes of sorghum revealed extensive hidden genomic variations be-tween cultivated and wild species [56]. For example, a pan-genome was generated for 13 genotypes of sorghum to explore the genetic variations between the cultivated and wild species. The developed pan-genome consists of core genes (58.8%), shell genes (37.9%), private genes (3.3%) and cloud genes (0.4%). Among these, core genes are found to be involved in RNA processing, reproductive system development, leaf development, seed development, cell differentiation, and chloroplast organization in sorghum [56]. They have also identified 19,359 and 1,47,899 presence and absence variants associated with sorghum domestications and grain-colour variation. In another study, the pan-genome of 176 genotypes of sorghum identified 18,898 variable genes associated with various stresses, 1,788 drought-responsive genes and 2.0 million SNPs [57]. Similarly, a pan-genome assembly of eleven pearl millet genotypes identified structural variations in endoplasmic reticulum-related genes associated with heat stress [58]. A total of 39,143 gene families, including 46.60–52.08% of core genes, 39.75–49.94% of dispensable genes and 0.73–8.73% of private genes, were obtained from the developed pan-genome of pearl millet. Additionally, 7,44,364 structural variations associated with heat-related genes were identified, including 306,679 presence variations, 315,905 absence variations, 2,177 in-versions, 91,852 copy number variations, and 27,751 translocations [58]. In minor millets, a pan genome sequence has been generated for foxtail millet using 110 genotypes (35 wild, 40 landraces, and 35 modern cultivated) [59]. The developed pan-genome of foxtail millet contains 73,528 gene families, of which 23.8%, 42.9%, 29.4% and 3.9% are core, soft core, dispensable and individual genes, respectively. In addition, 14,283 gene families involved in RNA capping, light response and specific metabolic processes were identified from the pan-genome of foxtail millet [59]. It is interesting to note that these gene families are not already present in the available Yugu1 reference genome. In the same study, approximately 2,02,884 non-redundant structural variations (1,07,151 insertions, 76,915 deletions, 18,455 translocations and 363 inversions) and 1,58,906 presence and absence variations associated with foxtail millet domestication and improvement were detected in the pan-genome of foxtail millet. Thirty-two proso millet high-quality pan-genomes contained 27,727 core, 8,288 softcore, 24,494 dispensable and 5,533 private gene families [60]. From the developed pan-genome assemblies 207,033 structural variations and 50,515 presence or absence variants (26,195 deletions and 24,320 insertions) related to 43 domestications and 31 agronomic traits of proso millet were identified. The developed millet pan-genome represents an important resource for millet improvement and gene discovery. Novel genes identified in the pan-genomes of millets can be reintroduced into high-yielding millets by implementing traditional plant breeding, genetic selection and transgenic approaches. Apart from the pan-genome, telomere-to-telomere genome has been developed for HYZ-T2T genotypes of sorghum to identify structural genes, transcription factors, and transporters involved in the biosynthetic pathways of tannins in the sorghum [61]. These millet pan-genomic resources will be useful for achieving the SDGs in developing coun-tries by accelerating the genetic gain in arid and semi-arid ecologies.

Q2. Many sections of the article do not reflect the most recent developments, such as QTLs, GWAS studies.

Ans. We have already listed all the available reports on major and minor millets, except sorghum. There are many reports of QTLs and GWAS studies related to sorghum. However, we have included the GWAS studies on sorghum in Table 4 based on available reports. We feel like sorghum has already gained popularity among millet and other plant researchers. As a result, we hope that this review article will compile and inform the plant research community about the resources available for other millets. Based on our knowledge, all other available reports on major and minor millets are properly discussed in the manuscript.

3. In Section 9, the genes discussed are predominantly derived from gene family analyses and omics datasets. I contend that the utility of these genes as references is markedly limited, and thus, they should not be emphasized as a principal component of the manuscript. Actually, remarkable progress has been made in the molecular biology of foxtail millet and sorghum in recent years, progress that this manuscript fails to acknowledge or incorporate.

Ans.  We agree with the reviewer's suggestions. In Section 9, we have included new contents on molecular biology studies of millets, such as functional characterization of genes through the CRISPR/Cas9 system based on the available reports on foxtail millet, tef, and sorghum, as shown below.

“In recent years, the genome editing tool CRISPR/Cas has gained popularity among plant science researchers to study the function of genes and generate stress-resistant and nutrient-rich plants. The CRISPR/Cas9 system has been widely applied in diverse plants, including millets [137]. This tool has been successfully implemented in sorghum, tef and foxtail millet. Knockout of the SEMIDWARF-1 gene in tef through the CRISPR/Cas9 system enabled the development of lodging-resistant varieties (dwarf plants) of tef [138]. Induced site-directed mutations in the kafirin genes of sorghum by CRISPR/Cas9 improved protein digestibility and vitreous endosperm [139,140]. Herbicide tolerant foxtail millet was developed by targeting two genes (acetolactate synthase (SiALS) and acetylcoenzyme A carboxylase (SiACC)) through CRISPR base editors (cytosine and adenosine base editors) [141]. Knockout of two carotenoid cleavage dioxygenase genes 8 (SbCCDa and SbCCDb) reduced or-obanchol production and parasite weed (in particular Striga) germination in sorghum [142]. Hence, further functional characterization of key genes from already available re-ports through genome editing approaches will help to enhance millet growth, which will support improving food availability in 2050.”

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments in the attachment.

Comments for author File: Comments.pdf

Author Response

General Comments: The authors, using numerous literature items, comprehensively presented the importance of genome sequencing for future food security. Before publishing your article, please consider the following points:

Ans. Thank you for your positive response and valuable comments to improve our manuscript. We have rectified all the errors as per your suggestions in the revised manuscript and responded properly to each question you raised below.

Q1. Line 39-40 “Millets’ production traditionally does not depend on artificial fertilizers, and most are unaffected by storage pests [3]” Are the authors really talking about pests?  See below. [3]Ceasar, S.A; Maharajan, T. The role of millets in attaining United Nation's sustainable developmental goals. Plants People Planet. 464 2022, 4, 345-349.

Ans. Thanks for indicating the error, we have changed the citations. The newly cited articles are mentioned below.

• Nagaraja, T. E., Nandini, C., Bhat, S., & Parveen, S. (2023). Artificial hybridization techniques in small millets—A review. Frontiers in Plant Science, 14, 1112117.
• Abioye, V. F., Babarinde, G. O., Ogunlakin, G. O., Adejuyitan, J. A., Olatunde, S. J., & Abioye, A. O. (2022). Varietal and processing influence on nutritional and phytochemical properties of finger millet: A review. Heliyon.

Q2. The seeds can be stored for more than 5 years without insect damage which makes it a most valuable crop in drought-prone areas of Africa (Latha et al., 2005)” A. Madhavi Latha, K. Venkateswara Rao, V. Dashavantha Reddy, Production of transgenic plants resistant to leaf blast disease in finger millet (Eleusine coracana (L.) Gaertn.), Plant Science, Volume 169, Issue 4, 2005, Pages 657-667, ISSN 0168-9452, https://0-doi-org.brum.beds.ac.uk/10.1016/j.plantsci.2005.05.009.

Ans. Thank you for your suggestion. However, we did not discuss on “finger millet seeds can be stored more than 5 years without insect damage” inside the manuscript. We have already developed disease resistant transgenic finger millet using antifungal protein-encoding gene (Ceasar and Ignacimuthu, 2012). We also identified QTL for several leaf blast resistance traits in finger millet using SSR markers through association mapping analysis (Ramakrishnan et al. 2016). Both references are already cited in the appropriate places.

• Ignacimuthu, S., & Ceasar, S. A. (2012). Development of transgenic finger millet (Eleusine coracana (L.) Gaertn.) resistant to leaf blast disease. Journal of Biosciences, 37, 135-147.
• Ramakrishnan, M., Antony Ceasar, S., Duraipandiyan, V., Vinod, K. K., Kalpana, K., Al-Dhabi, N. A., & Ignacimuthu, S. (2016). Tracing QTLs for leaf blast resistance and agronomic performance of finger millet (Eleusine coracana (L.) Gaertn.) genotypes through association mapping and in silico comparative genomics analyses. PLoS One, 11(7), e0159264.

Q3. “Since the improvement of finger millets for blast resistance – through conventional methods – is hampered by limited genetic variability, it is worthwhile to produce disease resistant transgenic plants using novel antifungal protein-encoding gene(s)

Ans: Thank you for your suggestion. We have included the sentences in appropriate places (L. Nos. 325-329) in the revised manuscript.

“Development of finger millet against blast and other diseases by conventional breeding methods is hampered by limited genetic variability. Hence, generating blast dis-ease-resistant finger millet using antifungal protein-encoding genes would be useful. The annotated genome sequence of finger millet could pave way to identify genes associated with blast and other diseases”.

Q4. Line 303 “with several traits in plants and millets.” - millet is also a plant

Ans. Thank you for your suggestions. We have removed the phrase “millets” in L. No. 412.

Q5 Line 425. “More than 30 thousand millet cultivars are globally available” - please indicate references

Ans. Thank you for your suggestion. We have created one suitable table for millet germplasm as per the reviewer 2’s suggestion. As per your suggestion, we have included suitable reference in L. No. 616.

• Vetriventhan, M., Azevedo, V. C., Upadhyaya, H. D., Nirmalakumari, A., Kane-Potaka, J., Anitha, S., ... & Tonapi, V. A. (2020). Genetic and genomic resources, and breeding for accelerating improvement of small millets: current status and future interventions. The Nucleus, 63, 217-239.

Reviewer 2 Report

Dear editor and colleagues,

I have cautiously read the submitted manuscript “Role of genome sequences of millets to strengthen food and nutritional scarcity for future generation”, submitted to Agriculture- mdpi.

It is a review article summarizing the progress of genomic progresses in the field of millets (proso, finger, barnyard, pearl etc). Although the authors have undeniably done a lot of work, it is my impression that the chosen subject has already been adequately addressed in millets’ literature.

As a result, I found a lot of correlations and overlapping to already published reviews. This, unfortunately, substantially reduces the novelty of the present manuscript and weakens its importance. Unless the authors incorporate data such as nutrients, evolution, ploidy of millets, germplasm resources, or something not extensively studied, this work is merely a repetition of already available/published knowledge.

Hence, based on the novelty findings of the paper, I regrettably have to propose rejection as it currently stands.

References

Ajeesh Krishna TP, Maharajan T, Ceasar SA. Improvement of millets in the post-genomic era. Physiol Mol Biol Plants. 2022 Mar;28(3):669-685. doi: 10.1007/s12298-022-01158-8. Epub 2022 Mar 29. PMID: 35465206; PMCID: PMC8986959.

Ceasar, A. Genome-editing in millets: current knowledge and future perspectives. Mol Biol Rep 49, 773–781 (2022). https://0-doi-org.brum.beds.ac.uk/10.1007/s11033-021-06975-w

Sood, S., Joshi, D.C., Chandra, A.K. et al. Phenomics and genomics of finger millet: current status and future prospects. Planta 250, 731–751 (2019). https://0-doi-org.brum.beds.ac.uk/10.1007/s00425-019-03159-6

Pooja Choudhary, Pooja Shukla, Mehanathan Muthamilarasan, Genetic enhancement of climate-resilient traits in small millets: A review, Heliyon, Volume 9, Issue 4, 2023, e14502, ISSN 2405-8440, https://0-doi-org.brum.beds.ac.uk/10.1016/j.heliyon.2023.e14502.

Sood S, Kumar A, Kalyana Babu B, Gaur VS, Pandey D, Kant L and Pattnayak A (2016) Gene Discovery and Advances in Finger Millet [Eleusine coracana (L.) Gaertn.] Genomics—An Important Nutri-Cereal of Future. Front. Plant Sci. 7:1634. doi: 10.3389/fpls.2016.01634.

Minor editing of English language required

Author Response

Q1. I have cautiously read the submitted manuscript “Role of genome sequences of millets to strengthen food and nutritional scarcity for future generation”, submitted to Agriculture- mdpi. It is a review article summarizing the progress of genomic progresses in the field of millets (proso, finger, barnyard, pearl etc). Although the authors have undeniably done a lot of work, it is my impression that the chosen subject has already been adequately addressed in millets’ literature. As a result, I found a lot of correlations and overlapping to already published reviews. This, unfortunately, substantially reduces the novelty of the present manuscript and weakens its importance. Unless the authors incorporate data such as nutrients, evolution, ploidy of millets, germplasm resources, or something not extensively studied, this work is merely a repetition of already available/published knowledge.

Ans. We agreed to the reviewer 2 suggestion. Some review articles related to the manuscript have already been published in various journals. However, the available review articles such as Sood et al. 2016 (doi: 10.3389/fpls.2016.01634.), Sood et al. 2019 (https://0-doi-org.brum.beds.ac.uk/10.1007/s00425-019-03159-6), Ceasar 2022 (https://0-doi-org.brum.beds.ac.uk/10.1007/s11033-021-06975-w)  and Pooja Choudhary, 2023 (https://0-doi-org.brum.beds.ac.uk/10.1016/j.heliyon.2023.e14502) have not yet documented the genomic resources of millets elaborately. They have very briefly described about the genomic resources of millets as one of the sections in their manuscripts. That is why we have proposed this review paper to millet researchers to know about the genetic and genomic resources of millets.  In addition, we have included several insightful sub-titles compared to existing review articles. We would like to thank the reviewer 2 for providing some more sub-titles to strengthen the manuscript. As per your suggestions, we have included genomic resources of millets, ploidy level and germplasm resources of millets, nutritional importance and health benefits of millets, and evolution of millets in the revised manuscript. We believe that the revised manuscript is suitable for publication in the MDPI-Agriculture.

Nutritional profile and health benefits of millets

Nutritional profile and health benefits of millets

Millets are nutritionally excellent because their grains are rich in proteins, minerals, flavonoids, polyphenols, and vitamins; therefore, they may offer multiple health benefits. About 80% of millet grains have long been an important part of the nutritious diet. Many research/review articles have already discussed the nutritional importance and health benefits of millets [16-18]. Millets are now considered “God's own cereal” due to their rich nutritional profile. Consuming millets in our daily diet raises the levels of proteins (especially adiponectin) that help protect against cardiovascular diseases [19]. Millets also contains higher amount of vitamin B3/niacin, which helps lower certain risk factors of heart diseases such as high cholesterol and triglycerides, and is effective in lowering oxi-dative stress [20]. Millet grains contain lowest carbohydrate than other cereals (especially rice) and so are highly recommended for people with type 2 diabetes [21]. Oxidative stress can cause various chronic diseases (neurodegenerative disorders, arthritis, and diabetes) [22]. A high-fat diet is also a risk factor for the development of dementia because it in-creases oxidative stress in the brain [23]. Millets are a good source of antioxidants, which can help support the body’s ability to fight oxidative stress, a factor in illness and aging [24]. Hence, consuming millets could decrease the risk of chronic diseases [24]. Millets are rich in phytochemicals (polyphenols, lignans, phytosterols, phyto-oestrogens, phytocya-nins) that help protect people from age-related degenerative diseases like diabetes, cancer etc [25]. Each millet has some unique nutritional properties that help improve human health. For example, sufficient amount of calcium is essential for bone health, blood vessel and muscular contractions, and to ensure proper nerve function [12]. Finger millet has a higher calcium content than all other millets, cereals and milk [17]; so finger millet is one of the best grain sources to improve/maintain proper calcium levels in humans [26]. Proso millet contains high lecithin which supports the neural health system [27]. Kodo millet contains high amount of potassium (>120mg/100 g), which helps reduce the abdominal cramps during the menstrual cycle [28,29]. Including pearl millet in our daily diet is an effective way to prevent iron deficiency anemia as its grains are rich in iron [13]. Overall, the consumption of millets reduces risk of heart disease, protects from diabetes, improves digestive system, lowers the risk of cancer, detoxifies the body, increases immunity in res-piratory health, increases energy levels and improves muscular and neural systems and are protective against several degenerative diseases.

Germplasm resources of millets

Germplasm resources are an essential strategic resource for continued progress in crop improvement for global food security, and nutrition. Many millet researchers have already discussed about the genetic resource of millets in various review articles [30,31]. The recent report on global millets conservation strategy indicates that more than 479,000 germplasm of sorghum and millets are conserved globally [32]. The millet germplasms are majorly conserved in Asian and African countries such as India, China, Japan, Kenya, Ethiopia, Uganda, and Zambia. It is noteworthy that developed countries such as US, Canada, France, Russia and Italy are also conserved millets germplasm [31]. The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, Tel-angana, India conserves a largest collection of millets (about 80,000 accessions of eight millets) which includes sorghum (42869 accessions), pearl millet (25537 accessions), fin-ger millet (7513 accessions), foxtail millet (1542 accessions), barnyard millet (749 accessions), kodo millet (665 accessions), little millet (473 accessions) and proso millet (849 ac-cessions) (https://genebank.icrisat.org/ accessed on March 2024). The ICARI-National Bu-reau of Plant Genetic Resources Institute (ICAR-NBPGRI), New Delhi, India conserves over 58,000 accessions of millet including sorghum (25669), pearl millet (8699), finger millet (11667), foxtail millet (4685), proso millet (1055), barnyard millet (2010), kodo millet (2404), little millet (2226), and browntop millet (44). (http://genebank.nbpgr.ernet.in/SeedBank/Default.aspx accessed on March 2024). The United States Department of Agriculture (USDA-ARS) gene bank conserved five millets germplasms (such as 1452 finger millet, 1314 pearl millet, 300 kodo millet, 212 little millet and 719 proso millet) (https://www.ars.usda.gov/southeast-area/griffin-ga/pgrcu/ ac-cessed on March 2024). Over the past 15 years, conservation of millets germplasm re-sources has become an important part of national and international research programs. However, number of germplasm available for small millets particularly little millet, kodo millet, barnyard millet, brown top millet, and other minor millets is very low [31]. Because researchers have not yet prioritized those millets and most of traditional genotypes have already disappeared due to the dominance of other cash crops.  Hence, collection and conservation of existing small millets is crucial before we lose them forever, which may help to support millets improvement globally.  

Evolution of millets

Millets are believed to be the ancient oldest domesticated crops. There is evidence that millets were cultivated in Asia and Africa over 5000 to 10000 years ago [33]. Archaeobotanical evidence has proved that foxtail millet and proso millet were first domesticated in China ~10500 calendar years before the present (cal. BP) [34]. Pearl millet has its origin in Africa and the earliest evidence of its cultivation is reported from Northeast Mali (4500 cal. BP) [36,37]. Kodo millet was originated in India and its domestication took place about 3000 years ago [28, 35]. Little millet was domesticated in several sites across India around 6400 cal. BP [38]. Hence, millets are not new to the world; they have always been a part of our staple food since ancient times. Due to the ability of millets to grow on marginal lands with low external inputs such as water, fertilizer, pesticides etc., farmers are trying to re-vive its cultivation and bring it back to the market.

Ploidy level of millets

The genome size, ploidy level, and chromosome number are of great importance for studying the evolution of millets and the development of the breeding program [39]. In addition, knowledge of a plant's genome size and ploidy status can provide clues about the mechanisms responsible for decreases or increases in genomic content along the evo-lutionary pathway [39]. The genome size and ploidy levels varied extensively among the millets. The ploidy level and chromosome number of each millet are provided in Table 1. Among the millets, pearl millet, kodo millet, and finger millet have the largest genome sizes. The genome size for little millet has not yet been estimated. Foxtail millet is a mem-ber of the subfamily Panicoideae and the tribe Paniceae, with chromosome numbers of 2n = 2x = 18 [40,41]. It is recognized as a diploid, but is closely related to many tetraploid and higher ploidy level species. Finger millet is an allotetraploid (2n = 4x = 36) that belongs to the Chloridoideae subfamily [42-44]. Barnyard millet is a hexaploid (2n = 6x = 54) and its belongs to the sub-family Panicoideae [45]. Three millets such as proso millet, kodo millet and little millet are tetraploid and their chromosome range is 36-40 [46,47] (Table 1).

Table 1. Details on ploidy level and genome size of millets based on the available reports

Reviewer 3 Report

Dear Editors

Millet could be an important crop in the future.

This manuscript present in a good way the molecular tools regarding agronomical characteristics. However, since a part of that information have been presented. In order to be a complete study about millets. I would like from authors to:

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add after line 395 a whole sentence (~0.5-1 pages) about molecular studies and nutrients.

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add a sentence (~0.5-1 pages) where a ranking of the more useful sources of germplasm and the most possible sources of germplasm will presented

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to add more information in sentence 6 i.e. about future prospects

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to add more information in conclusions.

Best Regards

Comments for author File: Comments.pdf

Author Response

General Comments: Millet could be an important crop in the future. This manuscript present in a good way the molecular tools regarding agronomical characteristics.

Q1. However, since a part of that information have been presented. In order to be a complete study about millets. add after line 395 a whole sentence (~0.5-1 pages) about molecular studies and nutrients.

Ans. Thank you for providing positive response to our manuscript. We have improved the manuscript as per your suggestions.

We have already discussed about the molecular studies (Sub headings 6-9). However, we have newly added the nutritional importance and health benefits of millets as per your suggestions.

Nutritional profile and health benefits of millets

Millets are nutritionally excellent because their grains are rich in proteins, minerals, flavonoids, polyphenols, and vitamins; therefore, they may offer multiple health benefits. About 80% of millet grains have long been an important part of the nutritious diet. Many research/review articles have already discussed the nutritional importance and health benefits of millets [16-18]. Millets are now considered “God's own cereal” due to their rich nutritional profile. Consuming millets in our daily diet raises the levels of proteins (especially adiponectin) that help protect against cardiovascular diseases [19]. Millets also contains higher amount of vitamin B3/niacin, which helps lower certain risk factors of heart diseases such as high cholesterol and triglycerides, and is effective in lowering oxidative stress [20]. Millet grains contain lowest carbohydrate than other cereals (especially rice) and so are highly recommended for people with type 2 diabetes [21]. Oxidative stress can cause various chronic diseases (neurodegenerative disorders, arthritis, and diabetes) [22]. A high-fat diet is also a risk factor for the development of dementia because it in-creases oxidative stress in the brain [23]. Millets are a good source of antioxidants, which can help support the body’s ability to fight oxidative stress, a factor in illness and aging [24]. Hence, consuming millets could decrease the risk of chronic diseases [24]. Millets are rich in phytochemicals (polyphenols, lignans, phytosterols, phyto-oestrogens, phytocyanins) that help protect people from age-related degenerative diseases like diabetes, cancer etc [25]. Each millet has some unique nutritional properties that help improve human health. For example, sufficient amount of calcium is essential for bone health, blood vessel and muscular contractions, and to ensure proper nerve function [12]. Finger millet has a higher calcium content than all other millets, cereals and milk [17]; so finger millet is one of the best grain sources to improve/maintain proper calcium levels in humans [26]. Proso millet contains high lecithin which supports the neural health system [27]. Kodo millet contains high amount of potassium (>120mg/100 g), which helps reduce the abdominal cramps during the menstrual cycle [28,29]. Including pearl millet in our daily diet is an effective way to prevent iron deficiency anemia as its grains are rich in iron [13]. Overall, the consumption of millets reduces risk of heart disease, protects from diabetes, improves digestive system, lowers the risk of cancer, detoxifies the body, increases immunity in respiratory health, increases energy levels and improves muscular and neural systems and are protective against several degenerative diseases.

Q2. add a sentence (~0.5-1 pages) where a ranking of the more useful sources of germplasm and the most possible sources of germplasm will presented

Ans. Thank you for your suggestion. We have included the germplasm resources of millets in the revised manuscript as per your suggestion.

Germplasm resources of millets

Germplasm resources are an essential strategic resource for continued progress in crop improvement for global food security, and nutrition. Many millet researchers have already discussed about the genetic resource of millets in various review articles [30,31]. The recent report on global millets conservation strategy indicates that more than 479,000 germplasm of sorghum and millets are conserved globally [32]. The millet germplasms are majorly conserved in Asian and African countries such as India, China, Japan, Kenya, Ethiopia, Uganda, and Zambia. It is noteworthy that developed countries such as US, Canada, France, Russia and Italy are also conserved millets germplasm [31]. The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, Telangana, India conserves a largest collection of millets (about 80,000 accessions of eight millets) which includes sorghum (42869 accessions), pearl millet (25537 accessions), fin-ger millet (7513 accessions), foxtail millet (1542 accessions), barnyard millet (749 accessions), kodo millet (665 accessions), little millet (473 accessions) and proso millet (849 ac-cessions) (https://genebank.icrisat.org/ accessed on March 2024). The ICARI-National Bureau of Plant Genetic Resources Institute (ICAR-NBPGRI), New Delhi, India conserves over 58,000 accessions of millet including sorghum (25669), pearl millet (8699), finger millet (11667), foxtail millet (4685), proso millet (1055), barnyard millet (2010), kodo millet (2404), little millet (2226), and brown-top millet (44). (http://genebank.nbpgr.ernet.in/SeedBank/Default.aspx accessed on March 2024). The United States Department of Agriculture (USDA-ARS) gene bank conserved five millets germplasms (such as 1452 finger millet, 1314 pearl millet, 300 kodo millet, 212 little millet and 719 proso millet) (https://www.ars.usda.gov/southeast-area/griffin-ga/pgrcu/ ac-cessed on March 2024). Over the past 15 years, conservation of millets germplasm re-sources has become an important part of national and international research programs. However, number of germplasm available for small millets particularly little millet, kodo millet, barnyard millet, brown top millet, and other minor millets is very low [31]. Because researchers have not yet prioritized those millets and most of traditional genotypes have already disappeared due to the dominance of other cash crops.  Hence, collection and conservation of existing small millets is crucial before we lose them forever, which may help to support millets improvement globally.  

Q3. to add more information in sentence 6 i.e. about future prospects. to add more information in conclusions.

Ans. As per your suggestion, we have included some insights in the conclusion and future prospects section.

“Maintaining millet genetic resources in seed banks and the conservation of the wild millet genotypes provide the genetic resources that are required for sustainable food production. More awareness should be given for collection and conservation of little millet, brown-top millet, tef, fonio millet, job’s tear, kodo millet, proso millet and barnyard millet. Because most of the traditional germplasms of those millets have already disappeared from the world. Improving the nutritional contents of each millet through nutritional transporter gene manipulation may help enhance the nutritional availability in seeds of millets. In addition, several differentially expressed genes and molecular markers have already been identified for major and minor millets through transcriptomic resources. Hence, millet re-searchers can try to use the transcriptomic resources of millets for identifying candidate genes and developing molecular markers for those millets without complete annotated genome sequences”.