Understanding the Effects of Growing Seasons, Genotypes, and Their Interactions on the Anthesis Date of Wheat Sown in North China
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Experimental Site and Growing Conditions
2.2. Experimental Design and Plant Material
2.3. Data Analysis
3. Results
3.1. Response of Wheat Anthesis to Growing Seasons
3.2. Response of Anthesis to Wheat Genotypes and Varieties Eras
3.3. Response of Wheat Anthesis to the GGI
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
- Slafer, G.A.; Whitechurch, E.M. Chapter 14. Manipulating wheat development to improve adaptation. In Application of Physiology in Wheat Breeding; Reynolds, M.P., Ortiz-Monasterio, J.I., McNab, A., Eds.; CIMMYT: Mexico City, Mexico, 2001; pp. 160–171. [Google Scholar]
- Craufurd, P.Q.; Wheeler, T.R. Climate change and the flowering time of annual crops. J. Exp. Bot. 2009, 60, 2529–2539. [Google Scholar] [CrossRef] [Green Version]
- Curtis, D.L. The Relation Between Yield and Date of Heading of Nigerian Sorghums. Exp. Agric. 1968, 4, 93–101. [Google Scholar] [CrossRef]
- Flohr, B.; Hunt, J.; Kirkegaard, J.; Evans, J.; Trevaskis, B.; Zwart, A.; Swan, A.; Fletcher, A.; Rheinheimer, B. Fast winter wheat phenology can stabilise flowering date and maximise grain yield in semi-arid Mediterranean and temperate environments. Field Crops Res. 2018, 223, 12–25. [Google Scholar] [CrossRef]
- Trnka, M.; Rötter, R.; Ruiz-Ramos, M.; Kersebaum, K.C.; Olesen, J.E.; Žalud, Z.; Semenov, M. Adverse weather conditions for European wheat production will become more frequent with climate change. Nat. Clim. Chang. 2014, 4, 637–643. [Google Scholar] [CrossRef]
- Wang, B.; Liu, D.L.; Asseng, S.; Macadam, I.; Yu, Q. Impact of climate change on wheat flowering time in eastern Australia. Agric. For. Meteorol. 2015, 209–210, 11–21. [Google Scholar] [CrossRef]
- Angus, J.F.; Mackenzie, D.H.; Morton, R.; Schafer, C.A. Phasic development in field crops II. Thermal and photoperiodic responses of spring wheat. Field Crops Res. 1981, 4, 269–283. [Google Scholar] [CrossRef]
- Challinor, A.; Watson, J.; Lobell, D.; Howden, S.; Smith, D.R.; Chhetri, N. A meta-analysis of crop yield under climate change and adaptation. Nat. Clim. Chang. 2014, 4, 287–291. [Google Scholar] [CrossRef]
- Jamil, M.; Ali, A.; Gul, A.; Ghafoor, A.; Napar, A.A.; Ibrahim, A.M.H.; Naveed, N.H.; Yasin, N.A.; Mujeeb-Kazi, A. Genome-wide association studies of seven agronomic traits under two sowing conditions in bread wheat. BMC Plant Biol. 2019, 19, 1–18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chen, C.; Fletcher, A.L.; Ota, N.; Flohr, B.; Lilley, J.M.; Lawes, R. Spatial patterns of estimated optimal flowering period of wheat across the southwest of Western Australia. Field Crops Res. 2020, 247, 107710. [Google Scholar] [CrossRef]
- Ottman, M.J.; Hunt, L.A.; White, J.W. Photoperiod and Vernalization Effect on Anthesis Date in Winter-Sown Spring Wheat Regions. Agron. J. 2013, 105, 1017–1025. [Google Scholar] [CrossRef] [Green Version]
- Díaz, A.; Zikhali, M.; Turner, A.S.; Isaac, P.; Laurie, D.A. Copy Number Variation Affecting the Photoperiod-B1 and Vernalization-A1 Genes Is Associated with Altered Flowering Time in Wheat (Triticum aestivum). PLoS ONE 2012, 7, e33234. [Google Scholar] [CrossRef] [Green Version]
- Christy, B.; Riffkin, P.; Richards, R.; Partington, D.; Acuña, T.B.; Merry, A.; Zhang, H.; Trevaskis, B.; O’Leary, G. An allelic based phenological model to predict phasic development of wheat (Triticum aestivum L.). Field Crops Res. 2020, 249, 107722. [Google Scholar] [CrossRef]
- Beales, J.; Turner, A.; Griffiths, S.; Snape, J.W.; Laurie, D.A. A Pseudo-ResponseRegulator is misexpressed in the photoperiod insensitive Ppd-D1a mutant of wheat (Triticum aestivum L.). Theor. Appl. Genet. 2007, 115, 721–733. [Google Scholar] [CrossRef] [PubMed]
- Worland, A.; Börner, A.; Korzun, V.; Li, W.; Petrovíc, S.; Sayers, E.J. The influence of photoperiod genes on the adaptability of European winter wheats. Euphytica 1998, 100, 385–394. [Google Scholar] [CrossRef]
- González, F.G.; Slafer, G.A.; Miralles, D.J. Pre-anthesis development and number of fertile florets in wheat as affected by photoperiod sensitivity genes Ppd-D1and Ppd-B1. Euphytica 2005, 146, 253–269. [Google Scholar] [CrossRef]
- Álvaro, F.; Isidro, J.; Villegas, D.; García Del Moral, L.F.; & Royo, C. Breeding effects on grain filling, biomass partitioning, and demobilization in Mediterranean durum wheat. Agron. J. 2008, 100, 361–370. [Google Scholar] [CrossRef]
- Distelfeld, A.; Li, C.; Dubcovsky, J. Regulation of flowering in temperate cereals. Curr. Opin. Plant Biol. 2009, 12, 178–184. [Google Scholar] [CrossRef] [Green Version]
- Trevaskis, B.; Hemming, M.N.; Dennis, E.S.; Peacock, W.J. The molecular basis of vernalization-induced flowering in cereals. Trends Plant Sci. 2007, 12, 352–357. [Google Scholar] [CrossRef]
- Yan, L.; Fu, D.; Li, C.; Blechl, A.; Tranquilli, G.; Bonafede, M.; Sanchez, A.; Valárik, M.; Yasuda, S.; Dubcovsky, J. The wheat and barley vernalization gene VRN3 is an orthologue of FT. Proc. Natl. Acad. Sci. USA 2006, 103, 19581–19586. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Gibbs, D.J.; Tedds, H.M.; Labandera, A.-M.; Bailey, M.; White, M.; Hartman, S.; Sprigg, C.; Mogg, S.L.; Osborne, R.; Dambire, C.; et al. Oxygen-dependent proteolysis regulates the stability of angiosperm polycomb repressive complex 2 subunit VERNALIZATION 2. Nat. Commun. 2018, 9, 1–11. [Google Scholar] [CrossRef]
- Zhang, X.K.; Xiao, Y.G.; Zhang, Y.; Xia, X.C.; Dubcovsky, J.; He, Z.H. Allelic Variation at the Vernalization Genes Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 in Chinese Wheat Cultivars and Their Association with Growth Habit. Crop Sci. 2008, 48, 458–470. [Google Scholar] [CrossRef] [Green Version]
- Flood, R.G.; Halloran, G.M. The influence of certain chromosomes of the hexaploid wheat cultivar Thatcher on time to ear emergence in Chinese Spring. Euphytica 1983, 32, 121–124. [Google Scholar] [CrossRef]
- Harris, F.A.J.; Eagles, H.A.; Virgona, J.M.; Martin, P.J.; Condon, J.R.; Angus, J.F. Effect of VRN1 and PPD1 genes on anthesis date and wheat growth. Crop Pasture Sci. 2017, 68, 195. [Google Scholar] [CrossRef]
- Dawit, T.; Wuletaw, T.; Muluken, B.; Tsegaye, D.; Tadesse, W.; Bayable, M. Genotype X environment interactions and grain yield stability of haricot bean varieties in Northwest Ethiopia. Sci. Res. Essays 2012, 7, 3487–3493. [Google Scholar] [CrossRef] [Green Version]
- Vaezi, B.; Pour-Aboughadareh, A.; Mohammadi, R.; Mehraban, A.; Hossein-Pour, T.; Koohkan, E.; Ghasemi, S.; Moradkhani, H.; Siddique, K. Integrating different stability models to investigate genotype × environment interactions and identify stable and high-yielding barley genotypes. Euphytica 2019, 215, 63. [Google Scholar] [CrossRef]
- Trethowan, R.M.; Van Ginkel, M.; Rajaram, S. Progress in Breeding Wheat for Yield and Adaptation in Global Drought Affected Environments. Crop Sci. 2002, 42, 1441–1446. [Google Scholar] [CrossRef]
- Zheng, B.; Chenu, K.; Chapman, S.C. Velocity of temperature and flowering time in wheat—Assisting breeders to keep pace with climate change. Glob. Chang. Biol. 2016, 22, 921–933. [Google Scholar] [CrossRef]
- Mohammadi, R.; Amri, A. Analysis of Genotype × Environment Interactions for Grain Yield in Durum Wheat. Crop Sci. 2009, 49, 1177–1186. [Google Scholar] [CrossRef]
- Chenu, K.; Deihimfard, R.; Chapman, S. Large-scale characterization of drought pattern: A continent-wide modelling approach applied to the Australian wheatbelt—Spatial and temporal trends. New Phytol. 2013, 198, 801–820. [Google Scholar] [CrossRef] [PubMed]
- Raju, N.S.; Senguttuve, P.; Voleti, S.; Prasad, A.H.; Bhadana, V.; Revathi, P.; Kemparaju, K.; Chandran, S.R.; Singh, A.K.; Rao, P.K.; et al. Stability Analysis of Flowering and Yield Traits to High Temperature Stress Adopting Different Planting Dates in Rice (O. sativa L.). Int. J. Agric. Res. 2006, 8, 137–148. [Google Scholar] [CrossRef] [Green Version]
- Shahriari, Z.; Heidari, B.; Dadkhodaie, A. Dissection of genotype × environment interactions for mucilage and seed yield in Plantago species: Application of AMMI and GGE biplot analyses. PLoS ONE 2018, 13, e0196095. [Google Scholar] [CrossRef] [Green Version]
- Bocianowski, J.; Warzecha, T.; Nowosad, K.; Bathelt, R. Genotype by environment interaction using AMMI model and estimation of additive and epistasis gene effects for 1000-kernel weight in spring barley (Hordeum vulgare L.). J. Appl. Genet. 2019, 60, 127–135. [Google Scholar] [CrossRef] [Green Version]
- Rincent, R.; Malosetti, M.; Ababaei, B.; Touzy, G.; Mini, A.; Bogard, M.; Martre, P.; Le Gouis, J.; Van Eeuwijk, F. Using crop growth model stress covariates and AMMI decomposition to better predict genotype-by-environment interactions. Theor. Appl. Genet. 2019, 132, 3399–3411. [Google Scholar] [CrossRef]
- Pask, A.J.D.; Pietragalla, J.; Mullan, D.M.; Reynolds, M.P. Physiological Breeding II: A Field Guide to Wheat Phenotyping; Cimmyt: Mexico City, Mexico, 2012. [Google Scholar]
- Guillemaut, P.; Maréchal-Drouard, L. Isolation of plant DNA: A fast, inexpensive, and reliable method. Plant Mol. Biol. Rep. 1992, 10, 60–65. [Google Scholar] [CrossRef]
- Fu, D.; Szűcs, P.; Yan, L.; Helguera, M.; Skinner, J.S.; Von Zitzewitz, J.; Hayes, P.M.; Dubcovsky, J. Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat. Mol. Genet. Genom. 2005, 273, 54–65. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Nishida, H.; Yoshida, T.; Kawakami, K.; Fujita, M.; Long, B.; Akashi, Y.; Laurie, D.A.; Kato, K. Structural variation in the 5′ upstream region of photoperiod-insensitive alleles Ppd-A1a and Ppd-B1a identified in hexaploid wheat (Triticum aestivum L.), and their effect on heading time. Mol. Breed. 2013, 31, 27–37. [Google Scholar] [CrossRef]
- Li, X.; Guo, T.; Mu, Q.; Li, X.; Yu, J. Genomic and environmental determinants and their interplay underlying phenotypic plasticity. Proc. Natl. Acad. Sci. USA 2018, 115, 6679–6684. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Gauch, H.G. Statistical Analysis of Yield Trials by AMMI and GGE. Crop Sci. 2006, 46, 1488–1500. [Google Scholar] [CrossRef]
- Ding, Y.; Ren, G.; Shi, G.; Gong, P.; Zheng, X.; Zhai, P.; Zhang, D.; Zhao, Z.; Wang, S.; Wang, H.; et al. China’s national assessment report on climate change (I): Climate change in China and the future trend. Climate Chang Research. 2007, 3, 1. [Google Scholar] [CrossRef]
- Asseng, S.; Foster, I.A.N.; Turner, N.C. The impact of temperature variability on wheat yields. Glob. Chang. Biol. 2011, 17, 997–1012. [Google Scholar] [CrossRef]
- Hegde, S.G.; Valkoun, J.; Waines, J.G. Genetic diversity in wild wheats and goat grass. Theor. Appl. Genet. 2000, 101, 309–316. [Google Scholar] [CrossRef]
- Xiao, D.; Tao, F. Contributions of cultivar shift, management practice and climate change to maize yield in North China Plain in 1981–2009. Int. J. Biometeorol. 2016, 60, 1111–1122. [Google Scholar] [CrossRef]
- Yang, F.P.; Zhang, X.K.; Xia, X.C.; Laurie, D.A.; Yang, W.X.; He, Z.H. Distribution of the photoperiod insensitive Ppd-D1a allele in Chinese wheat cultivars. Euphytica 2009, 165, 445–452. [Google Scholar] [CrossRef]
- Cao, W.; Liu, S.; Yang, Q.; Zhang, W. Characteristics of vernalization gene and photoperiod gene and their relationship with winter hardness revealed by STS markers. Mol. Plant Breed. 2016, 14, 117–124. [Google Scholar]
- Boden, S.A.; Cavanagh, C.; Cullis, B.R.; Ramm, K.; Greenwood, J.; Jean, F.E.; Trevaskis, B.; Swain, S.M. Ppd-1 is a key regulator of inflorescence architecture and paired spikelet development in wheat. Nat. Plants 2015, 1, 14016. [Google Scholar] [CrossRef] [PubMed]
- Cockram, J.; Jones, H.; Leigh, F.; O’Sullivan, D.; Powell, W.; Laurie, D.A.; Greenland, A.J. Control of flowering time in temperate cereals: Genes, domestication, and sustainable productivity. J. Exp. Bot. 2007, 58, 1231–1244. [Google Scholar] [CrossRef] [PubMed]
- Snape, J.; Butterworth, K.; Whitechurch, E.; Worland, A. Waiting for fine times: Genetics of flowering time in wheat. Euphytica 2001, 119, 185–190. [Google Scholar] [CrossRef]
- Eagles, H.A.; Cane, K.; Kuchel, H.; Hollamby, G.J.; Vallance, N.; Eastwood, R.F.; Gororo, N.N.; Martin, P.J. Photoperiod and vernalization gene effects in southern Australian wheat. Crop Pasture Sci. 2010, 61, 721–730. [Google Scholar] [CrossRef]
- Shcherban, A.B.; Börner, A.; Salina, E.A. Effect ofVRN-1andPPD-D1genes on heading time in European bread wheat cultivars. Plant Breed. 2015, 134, 49–55. [Google Scholar] [CrossRef]
- Kim, D.-H.; Doyle, M.R.; Sung, S.; Amasino, R.M. Vernalization: Winter and the Timing of Flowering in Plants. Annu. Rev. Cell Dev. Biol. 2009, 25, 277–299. [Google Scholar] [CrossRef] [Green Version]
- Kumar, S.; Sharma, V.; Chaudhary, S.; Tyagi, A.; Mishra, P.; Priyadarshini, A.; Singh, A. Genetics of flowering time in bread wheat Triticum aestivum: Complementary interaction between vernalization-insensitive and photoperiod-insensitive mutations imparts very early flowering habit to spring wheat. J. Genet. 2012, 91, 33–47. [Google Scholar] [CrossRef] [PubMed]
- Daojie, S. Breeding photosensitivity enhanced wheat varieties to deal with the losses caused by climate change. J. Anhui Agric. Sci. 2007, 35, 10642. [Google Scholar] [CrossRef]
- Hill, C.B.; Li, C. Genetic Architecture of Flowering Phenology in Cereals and Opportunities for Crop Improvement. Front. Plant Sci. 2016, 7, 1906. [Google Scholar] [CrossRef] [Green Version]
- Gabriel, K.R. The biplot graphic display of matrices with application to principal component analysis. Biometrika 1971, 58, 453–467. [Google Scholar] [CrossRef]
- Geng, X.; Wang, F.; Ren, W.; Hao, Z. Climate Change Impacts on Winter Wheat Yield in Northern China. Adv. Meteorol. 2019, 2019, 1–12. [Google Scholar] [CrossRef] [Green Version]
- Fu, G.; Charles, S.P.; Yu, J.; Liu, C. Decadal Climatic Variability, Trends, and Future Scenarios for the North China Plain. J. Clim. 2009, 22, 2111–2123. [Google Scholar] [CrossRef]
- Bai, H.; Tao, F.; Xiao, D.; Liu, F.; Zhang, H. Attribution of yield change for rice-wheat rotation system in China to climate change, cultivars and agronomic management in the past three decades. Clim. Chang. 2015, 135, 539–553. [Google Scholar] [CrossRef]
- Liu, Y.; Wang, E.; Yang, X.; Wang, J. Contributions of climatic and crop varietal changes to crop production in the North China Plain, since 1980s. Glob. Chang. Biol. 2010, 16, 2287–2299. [Google Scholar] [CrossRef]
- Li, K.; Yang, X.; Tian, H.; Pan, S.; Liu, Z.; Lu, S. Effects of changing climate and cultivar on the phenology and yield of winter wheat in the North China Plain. Int. J. Biometeorol. 2016, 60, 21–32. [Google Scholar] [CrossRef]
- White, J.W.; Herndl, M.; Hunt, L.A.; Payne, T.S.; Hoogenboom, G. Simulation-Based Analysis of Effects of Vrn and Ppd Loci on Flowering in Wheat. Crop Sci. 2008, 48, 678–687. [Google Scholar] [CrossRef]
- Dixon, J.; Braun, H.-J.; Kosina, P.; Crouch, J. Wheat Facts and Futures; CIMMYT: Mexico City, Mexico, 2009. [Google Scholar]
Genotype | Photoperiod and Vernalization Genotype | Percentage (%) † |
---|---|---|
A1B1 | Ppd-A1a + Ppd-D1a + Vrn-D1 | 1.0 |
A1B2 | Ppd-A1a + Ppd-D1a + vrn-D1 | 4.0 |
A2B2 | Ppd-A1a + Ppd-D1b + vrn-D1 | 4.0 |
A3B1 | Ppd-A1b + Ppd-D1a + Vrn-D1 | 7.0 |
A3B2 | Ppd-A1b + Ppd-D1a + vrn-D1 | 58.0 |
A4B1 | Ppd-A1b + Ppd-D1b + Vrn-D1 | 6.0 |
A4B2 | Ppd-A1b + Ppd-D1b + vrn-D1 | 20.0 |
Growing Seasons | Source of Variance | Sum Sq | Mean Sq | F Value | Pr > F |
---|---|---|---|---|---|
16Y | Ppd-A1 | 76.8 | 76.8 | 8.3 | 0.004 |
Ppd-D1 | 2368.7 | 2368.7 | 256.7 | <0.001 | |
Vrn-D1 | 40.7 | 40.7 | 4.4 | 0.037 | |
Ppd-A1:Ppd-D1 | 3.4 | 3.4 | 0.4 | 0.546 | |
Ppd-A1:Vrn-D1 | 2.1 | 2.1 | 0.2 | 0.635 | |
Ppd-D1:Vrn-D1 | 52.1 | 52.1 | 5.7 | 0.018 | |
17Y | Ppd-A1 | 13.7 | 13.7 | 1.3 | 0.249 |
Ppd-D1 | 1003.7 | 1003.7 | 98 | <0.001 | |
Vrn-D1 | 9.5 | 9.5 | 0.9 | 0.336 | |
Ppd-A1:Ppd-D1 | 20.6 | 20.6 | 2 | 0.158 | |
Ppd-A1:Vrn-D1 | 42.8 | 42.8 | 4.2 | 0.042 | |
Ppd-D1:Vrn-D1 | 73.5 | 73.5 | 7.2 | 0.008 | |
18Y | Ppd-A1 | 42.2 | 42.2 | 5.2 | 0.024 |
Ppd-D1 | 1877.2 | 1877.2 | 230.2 | <0.001 | |
Vrn-D1 | 9.8 | 9.8 | 1.2 | 0.274 | |
Ppd-A1:Ppd-D1 | 2.3 | 2.3 | 0.3 | 0.596 | |
Ppd-A1:Vrn-D1 | 12.2 | 12.2 | 1.5 | 0.222 | |
Ppd-D1:Vrn-D1 | 91.9 | 91.9 | 11.3 | 0.001 |
Source of Variance | df | Sum of Squares | Fischer’s Ratio | % of Variance |
---|---|---|---|---|
Growing seasons (S) | 2 | 1047.7 | 347.8 *** | 66.3 |
Genotype (G) | 6 | 345.5 | 99.1 *** | 21.9 |
S:G | 12 | 158.9 | 11.6 *** | 10.1 |
IPCA1 | 7 | 150.3 | 35.2 *** | 94.6 |
IPCA2 | 5 | 8.6 | 2.8 * | 5.4 |
Residuals | 36 | 22.0 | 1.4 |
Variety Eras | Source of Variance | Df | Sum of Squares | Fischer’s Ratio | % of Variance |
---|---|---|---|---|---|
1940s | Growing seasons (S) | 2 | 288.8 | 212.5 *** | 25.0 |
Genotype (G) | 3 | 816.4 | 333.3 *** | 70.6 | |
S:G | 6 | 37.3 | 7.6 *** | 3.2 | |
Residuals | 18 | 14.7 | 1.2 | ||
1950s | Growing seasons (S) | 2 | 645.1 | 265.3 *** | 93.4 |
Genotype (G) | 3 | 28.9 | 31.6 *** | 4.2 | |
S:G | 6 | 11.1 | 6.1 ** | 1.6 | |
Residuals | 18 | 5.5 | 0.8 |
Gene | Allele | Effect | Reference | |
---|---|---|---|---|
Vernalization gene | Vrn-D1 | Vrn-D1 (Spring growth habit) | Promoted flowering | [23] |
vrn-D1 (Winter growth habit) | Delayed flowering | |||
Photoperiod genes | Ppd-A1 | Ppd-A1a (Photoperiod-insensitive gene) | Promoted flowering | [17] |
Ppd-A1b (Photoperiod-sensitive gene) | Delayed flowering | |||
Ppd-D1 | Ppd-D1a (Photoperiod-insensitive gene) | Promoted flowering | ||
Ppd-D1b (Photoperiod-sensitive gene) | Delayed flowering |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Li, Z.; Zheng, B.; He, Y. Understanding the Effects of Growing Seasons, Genotypes, and Their Interactions on the Anthesis Date of Wheat Sown in North China. Biology 2021, 10, 955. https://doi.org/10.3390/biology10100955
Li Z, Zheng B, He Y. Understanding the Effects of Growing Seasons, Genotypes, and Their Interactions on the Anthesis Date of Wheat Sown in North China. Biology. 2021; 10(10):955. https://doi.org/10.3390/biology10100955
Chicago/Turabian StyleLi, Ziwei, Bangyou Zheng, and Yong He. 2021. "Understanding the Effects of Growing Seasons, Genotypes, and Their Interactions on the Anthesis Date of Wheat Sown in North China" Biology 10, no. 10: 955. https://doi.org/10.3390/biology10100955