Assessing morpho-chemical and genetic diversity in acid limes germplasm using microsatellite markers

Arooge FATIMA; Muhammad USMAN; Bilquees FATIMA; Iqrar Ahmad RANA

doi:10.15835/nbha53314566

Authors

Arooge FATIMA University of Agriculture, Plant Tissue Culture Cell (PTCC), Institute of Horticultural Sciences, Faisalabad 38040 (PK)
Muhammad USMAN University of Agriculture, Plant Tissue Culture Cell (PTCC), Institute of Horticultural Sciences, Faisalabad 38040 (PK)
Bilquees FATIMA University of Agriculture, Plant Tissue Culture Cell (PTCC), Institute of Horticultural Sciences, Faisalabad 38040 (PK)
Iqrar Ahmad RANA University of Agriculture, Center of Agricultural Biochemistry and Biotechnology (CABB), Faisalabad 38040 (PK)

DOI:

https://doi.org/10.15835/nbha53314566

Keywords:

fruit breeding, germplasm, kagzi lime, molecular markers, ploidy, SSR

Abstract

Acid limes are highly nutraceutical and have great market demand. In Pakistan, two lime varieties are cultivated including ‘Mexican lime’ and ‘Eustis limequat’. Hence, other potential varieties were characterized for morphogenetic variability. Leaf shape was elliptic in ‘Mexican lime’ and ‘Eustis limequat’ and obcordate in ‘Tahiti lime’, ‘Seedless lime’ and ‘Tavares limequat’ while the leaf area was greater in ‘Seedless lime’. ‘Tavares limequat’ developed the heaviest fruit (74.55 g) with greater fruit length while fruit diameter was higher in ‘Seedless lime’. Juice weight and juice percentage were greater in ‘Tavares limequat’ and ‘Seedless lime’ respectively. ‘Tahiti lime’, ‘Seedless lime’ and ‘Tavares limequat’ were found as a seedless variety. Titratable acidity was higher in ‘Eustis limequat’ while sugar to acid ratio was lower in ‘Eustis limequat’, ‘Mexican lime’ and ‘Seedless lime’ varieties. Simple sequence repeat (SSR) markers analysis revealed great genetic diversity among populations. Maximum alleles (9-11) were amplified by using CAC23 and CTT01 markers which showed higher discrimination and heterozygosity. CAC23 and CTT01 markers effectively amplified three alleles in triploids (‘Tahiti lime’ and ‘Seedless lime’), a single allele in diploids (‘Mexican lime’, ‘Eustis limequat’) and two alleles, including a unique allele in diploid ‘Tavares limequat’. Cluster analysis bunched closely related genotypes into two groups, STRUCTURE analysis confirmed the admixture nature of all genotypes and ‘Tavares limequat’ was found to be the most divergent. Conclusively, selected SSR markers effectively discriminated among diploid and polyploid acid limes and may be extended to other citrus species as well. These findings could be helpful for the selection and conservation of heterozygous material and utilization in breeding and germplasm enhancement programs.

References

Amom T, Nongdam P (2017). The use of molecular marker methods in plants: a review. International Journal of Current Research and Review 9:1-7. https://doi.org/10.7324/ijcrr.2017.9171

Andrade-Rodríguez M, Villegas-Monter A, Carrillo-Castañeda G, García-Velázquez A (2004). Polyembryony and identification of Volkamerian lemon zygotic and nucellar seedlings using RAPD. Pesquisa Agropecuária Brasileira 39:551-559. https://doi.org/10.1590/s0100-204x2004000600006

Atak A, Aydin B, Kahraman KA (2014). Sex determination of kiwifruit with molecular markers. Acta Horticulture 1048:197-203. https://doi.org/10.17660/ActaHortic.2014.1048.24

Barkley NA, Roose ML, Krueger RR, Federici CT (2006). Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs). Theoretical and Applied Genetics 112:1519-1531. https://doi.org/10.1007/s00122-006-0255-9

Breto MP, Ruiz C, Pina JA, Asins MJ (2001). The diversification of Citrus clementina Hort. ex Tan, a vegetatively propagated crop species. Molecular Phylogenetic and Evolution 21:285-293. https://doi.org/10.1006/mpev.2001.1008

Canan I, Gündoğdu M, Seday U, Oluk CA, Karaşahin, ZK, Eroğlu EC, Yazici E, ÜNLÜ M (2016). Determination of antioxidant, total phenolic, total carotenoid, lycopene, ascorbic acid, and sugar contents of Citrus species and mandarin hybrids. Turkish Journal of Agriculture and Forestry 40:894-899. https://doi.org/10.3906/tar-1606-83

Chesnokov YV, Kosolapov VM, Savchenko IV (2020). Morphological genetic markers in plants. Russian Journal of Genetics 56:1406-1415. https://doi.org/10.1134/s1022795420120042

Curk F, Garcia-Lor A, Luro F, Snoussi-Trifa H, Froelicher Y, Ancillo G, Navarro L, Ollitrault P (2015). New insights on limes and lemons origin from nuclear and cytoplasmic markers genotyping and targeted nuclear gene sequencing. Acta Horticulture 1065:135-46. https://doi.org/10.17660/actahortic.2015.1065.13

Curk F, Ollitrault F, Garcia-Lor A, Luro F, Navarro L, Ollitrault (2016). Phylogenetic origin of times and lemons revealed by cytoplasmic and nuclear markers. Annals of Botany 117:565-83. https://doi.org/10.1093/aob/mcw005

Doyğaci Y, Atak A (2024). Analysis of genetic giversity of some Vitis labrusca and Vitis spp. with molecular markers. Anadolu Journal of Agricultural Sciences 39:607-621. https://doi.org/10.7161/omuanajas.1475187

Dubey AK, Sharma RM, Awasthi OP, Srivastav M, Sharma N (2016). Genetic diversity in lime (Citrus aurantifolia Swing.) and lemon (Citrus limon (L.) Burm.) based on quantitative traits in India. Agroforestry System 90:447-456. https://doi.org/10.1007/s10457-015-9867-x

Elameen A, Fjellheim S, Larsen A, Rognli OA, Sundheim L, Msolla S, Masumba E, Mtunda K, Klemsdal SS (2008). Analysis of genetic diversity in a sweet potato (Ipomoea batatas L.) germplasm collection from Tanzania as revealed by AFLP. Genetic Resources and Crop Evolution 55:397-408. https://doi.org/10.1007/s10722-007-9247-0

Ellegren H (2004). Microsatellites: simple sequences with complex evolution. Nature Reviews Genetics 5:435-445. https://doi.org/10.1038/nrg1348

FAOSTAT (2023) Food and Agriculture Organization of the United Nations Database. Retrieved in 2025 March 20 from http://faostat.fao.org/default.aspx

Fatima B, Usman M, Khan MS, Khan IA, Khan MM (2015). Identification of Citrus polyploids using chromosome counts, morphological and SSR markers. Pakistan Journal of Agricultural Sciences 52:107-114. http://pakjas.com.pk/papers/2395.pdf

Fattahi J, Hamidoghli Y, Fotouhi R, Ghasemnejad M, Bakhshi D (2004). Assessment of fruit quality and antioxidant activity of three citrus species during ripening. South Western Journal of Horticulture Biology and Environment 2:113-128. http://anucraiova.3x.ro/swjhbe/index.html

Garcia-Lor A, Curk F, Snoussi-Trifa H, Morillon R, Ancillo G, Luro F, Navarro L Ollitrault P (2013). A nuclear phylogenetic analysis: SNPs, indels and SSRs deliver new insights into the relationships in the ‘true citrus fruit trees’ group (Citrinae butaceae) and the origin of cultivated species. Annals of Botany 111:1-19. https://doi.org/10.1093/aob/mcs227

Gill K, Kumar P, Sharma V, Negi S, Sharma M, Sharma R, Joshi AK (2024). Unraveling phenotypic ambiguities of kagzi and ornamental lime accessions: a comprehensive exploration through morpho-biochemical and DNA marker profiling. Genetic Resources and Crop Evolution 71:3765-3790. https://doi.org/10.1007/s10722-024-01862-2

Giusti MM, Wrolstad RE (2001). Characterization and measurement of anthocyanins by UV‐visible spectroscopy. Current Protocols in Food Analytical Chemistry https://doi.org/10.1002/0471142913.faf0102s00

Golein B, Talaie A, Zamani Z, Moradi B (2006). Development and characterization of new microsatellite loci from lemon (Citrus limon). International Journal of Agriculture and Biology 8:172-174.

Gorinstein S, Martı́n-Belloso O, Park YS, Haruenkit R, Lojek A, Ĉı́ž M, Caspi A, Libman I, Trakhtenberg S (2001). Comparison of some biochemical characteristics of different citrus fruits. Food Chemistry 74:309-315. https://doi.org/10.1016/s0308-8146(01)00157-1

Hortwitz W (1960). Official and tentative methods of analysis. Association of Official Agriculture Chemists Washington D.C. https://doi.org/10.5962/bhl.title.44636

Hussein EH, Abd-alla SM, Awad NA, Hussein MS (2003). Genetic analysis in some Citrus accessions using microsatellites and AFLP-based markers. Arab Journal of Biotechnology 6:80-201. https://doi.org/10.21608/arjb.2003.434336

IPGRI (1999). Descriptors for Citrus. International Plant Genetic Resources Institute, Rome, Italy. ISBN 92-9043-425-2.

Jabeen S, Saif R, Haq R, Hayat A, Naz S (2023). Whole-genome sequencing and variant discovery of Citrus reticulata “Kinnow” from Pakistan. Functional and Integrative Genomics. 23:227. https://doi.org/10.1101/2022.12.07.519411

Kaur S, Singh H, Jawandha SK (2023). Elucidating the yield and quality response of lime and lemon under different spacings in sub-tropical conditions. Erwerbs-Obstbau 65:2575-2583. https://doi.org/10.1007/s10341-023-00957-x

Ketsa S (1998). Effects of fruit size on juice content and chemical composition of tangerine. Journal of Horticulture Science 63:171-174. https://doi.org/10.1080/14620316.1988.11515843

Khan IA, Awan FS, Ahmad A, Khan AA (2004). A modified mini-prep method for economical and rapid extraction of genomic DNA in plants. Plant Molecular Biology Reporter 22:89-89. https://doi.org/10.1007/bf02773355

Khan IA, Kender WJ (2007). Citrus breeding. In: Khan IA (Ed). Citrus genetics, breeding and biotechnology. CAB International, Wallingford, UK pp. 1-8. https://doi.org/10.1017/s0014479708006820

Kijas JMH, Thomas MR, Fowler JCS, Roose ML (1997). Integration of trinucleotide microsatellites into a linkage map of Citrus. Theoretical and Applied Genetics 94:701-706. https://doi.org/10.1007/s001220050468

Lado J, Rodrigo MJ, Zacarías L (2014). Maturity indicators and citrus fruit quality. Stewart Postharvest Review 10:1-6.

Lux PE, Carle R, Zacarías L, Rodrigo MJ, Schweiggert RM, Steingass CB (2019). Genuine carotenoid profiles in sweet orange [Citrus sinensis (L.) Osbeck cv. Navel] peel and pulp at different maturity stages. Journal of Agriculture and Food Chemistry 67:13164-13175. https://doi.org/10.1021/acs.jafc.9b06098

Moukoumbi YD, Sie M, Vodouhe R, N’dri B, Toulou B, Ogunbayo SA, Ahanchede A (2011) Assessing phenotypic diversity of interspecific rice varieties using agro-morphological characterization. Journal of Plant Breeding and Crop Science 3:74-86. http://www.academicjournals.org/jpbcs

Mukhopadhyay S (2010). Citrus production technology. Citrus production, post harvest, disease and pest management. (1st ed). CRC Press, Boca Raton p 290. https://doi.org/10.1201/9781482280210-12

Munankarmi NN, Rana N, Bhattarai T, Shrestha RL, Joshi BK, Baral, Hrestha S (2018). Characterization of the genetic diversity of acid lime (Citrus aurantifolia (Christm.) Swingle) cultivars of eastern Nepal using inter-simple sequence repeat markers. Plants 7:46. https://doi.org/10.3390/plants7020046

Munankarmi NN, Rana N, Joshi BK, Bhattarai T, Chaudhary S, Baral B, Shrestha S (2023). Characterization of the genetic diversity of Citrus species of Nepal using simple sequence repeat (SSR) markers. South African Journal of Botany 156:192-201. https://doi.org/10.1016/j.sajb.2023.03.014

Munankarmi NN, Shrestha RL, Rana N, Shrestha JKC, Shrestha S, Koirala R (2014). Genetic diversity assessment of acid lime (Citrus aurantifolia Swingle) landraces of eastern Nepal using RAPD markers. International Journal of Applied Science and Biotechnology 2:315-327. https://doi.org/10.3126/ijasbt.v2i3.10950

Ogunkanmi LA, Ogundipe OT, Fatokun CA (2008). Genetic diversity in wild relatives of Cowpea (Vigna unguiculata) as revealed by simple sequence repeat markers. Food Agriculture and Environment 6:264-268.

Pardo E, Hernández K, Cavadía T (2023). Molecular characterization of guava Psidium guajava in Cereté, Córdoba, Colombia. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 51(3):13305. https://doi.org/10.15835/nbha51313305

Paudyal KP and Haq N (2008). Variation of pomelo (Citrus grandis (L.) Osbeck) in Nepal and participatory selection of strains for further improvement. Agroforestry System 72:195-204. https://doi.org/10.1007/s10457-007-9088-z

Peakall R, Smouse P (2012). GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research - an update. Bioinformatics 28:2537-2539. https://doi.org/10.1093/bioinformatics/bts460

Prasanna VSSV, Madhavi M, Lakshmi LM, Rajasekharam T, Amaravathi Y, Krishna KU (2023). Assessment of variability in fruit, yield and biochemical characters of acid lime (Citrus aurantifolia Swingle) germplasm. Journal of Pharmaceutical Innovation 12:4232-4238.

Raheb S, Ghasemnezhad M, Golain B, Golmohammadi M, Sabori A (2019). Investigation of polymorphism in different acid lime (Citrus aurantifolia Swingle) genotypes with free witches’ broom disease by molecular markers of SSR and ISSR. Iranian Journal of Horticulture Sciences 50:1-11.

Rana MA, Usman M, Fatima B, Fatima A, Rana IA, Rehman W, Shoukat D (2020). Prospects of mutation breeding in grapefruit (Citrus paradisi Macf.). Journal of Horticulture Science and Technology 3:31-35. https://doi.org/10.46653/jhst20030231

Rouiss H, Bakry F, Froelicher Y, Navarro L, Aleza P, Ollitrault P (2018). Origin of C. latifolia and C. aurantiifolia triploid limes: the preferential disomic inheritance of doubled-diploid ‘Mexican’ lime is consistent with an interploid hybridization hypothesis. Annals of Botany 121:571-585. https://doi.org/10.1093/aob/mcx179

Ruck JA (1963). Chemical methods for analysis of fruit and vegetable products. Canada Department of Agriculture, Research Branch, Ottawa. https://doi.org/10.5962/bhl.title.58896

Sadasivam S. and Manickam A (1992). Biochemical Methods for Agricultural Sciences. Wiley Eastern Limited, New Delhi, India.

Sharafi AA, Abkenar AA, Sharafi A, Masaeli M (2016). Genetic variation assessment of acid lime accessions collected from south of Iran using SSR and ISSR molecular markers. Physiology and Molecular Biology of Plants 22:87-95. https://doi.org/10.1007/s12298-016-0336-4

Shidfar M, Akkurt M, Atak A, Ergül A, Söylemezoğlu G (2019). Evaluation of grapevine resistance to downy and powdery mildew in ‘Regent’ × ‘Boğazkere’ hybrid population segregating for resistance genes. Acta Scientiarum Polonorum Hortorum Cultus 18:181-188. https://doi.org/10.24326/asphc.2019.1.18

Sokal RR, Michener CD (1958). A statistical method for evaluating systematic relationships. University of Kansas Science Bulletin 38:1409-1438. https://doi.org/10.2317/jkes120710.1

Steel RGD, Torrie JH, Dicky DA (1997). Principles and procedures of statistics: A biological approach. McGraw Hill Book Co. (3rd ed), New York. https://doi.org/10.1002/bimj.19620040313

USDAFAS (2025). Citrus: World Market and Trade. United States Department of Agriculture Foreign Agricultural Service. United States. Retrieved in 2025 September 1 from https://www.fas.usda.gov/data/citrus-world-markets-and-trade

Usman M, Khan MM, Al-Yahyai, Fatima B (2019). Lime breeding: A way forward. In: Usman M, Khan MM, Al-Yahyai, Fatima B (Eds). Achieving sustainable cultivation of tropical fruits. Burleigh Dodds Science Publishing pp. 139-182. https://doi.org/10.19103/as.2019.0054.09

Usman M, Rehman W, Fatima B, Shahid M, Saggu AH, Rana MA, Fatima A (2020a). Fruit quality assessment in pigmented grapefruit (Citrus paradisi Macf.) for varietal diversification. Pakistan Journal of Agricultural Sciences 57:1029-1034.

Usman M, Zaman Q, Fatima B, Rana IA, Awan FS (2020b). Morpho-chemical diversity and RAPD fingerprinting in white flesh guava cultivars. Journal of Animal and Plant Sciences 30(2):398-409. https://doi.org/10.36899/JAPS.2020.2.0034

Varshney RK, Graner A, Sorrell ME (2005). Genomics-assisted breeding for crop improvement. Trends in Plant Sciences 10:621-630. https://doi.org/10.1016/j.tplants.2005.10.004

Vincent C, Morillon R, Arbona V, Gómez-Cadenas A (2020). Citrus in changing environments. In: Talon M, Caruso M, Gmitter FGJr (Eds). The Genus Citrus. Elsevier pp. 271-289. https://doi.org/10.1016/b978-0-12-812163-4.00013-9

Wang X, Xu Y, Zhang S (2017). Genomic analysis of primitive, wild and cultivated Citrus provides insights into asexual reproduction. Nature Genetics 49:665-722. https://doi.org/10.1038/ng.3839

Watanabe M, Yamamoto T, Ohara M, Nishitani C, Yahata S (2008). Cultivar differentiation identified by SSR markers and the application for polyploid loquat plants. Journal of Japanese Society and Horticultural Sciences 77:388-394. https://doi.org/10.2503/jjshs1.77.388

Yildirim Z, Atak A, Akkurt M (2019). Determination of downy and powdery mildew resistance of some Vitis spp. Ciência e Técnica Vitivinícola 34:15-24. https://doi.org/10.1051/ctv/20193401015

Yildiz E, Kaplankiran M, Demirkeser H, Uzun A Toplu C (2013). Identification of zygotic and nucellar individuals produced from several citrus crosses using SSRs markers. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 41:478-484. https://doi.org/10.15835/nbha4129037

Zamani Z, Adabi M, Khadivi-Khub A (2013). Comparative analysis of genetic structure and variability in wild and cultivated pomegranates as revealed by morphological variables and molecular markers. Plant System Evolution 299:1967-1980. https://doi.org/10.1007/s00606-013-0851-5

Zandkarimi H, Talaie A, Fatahi R, Jaime A, da Silva, T (2011). Evaluation of some lime and lemon accessions by using morphological characterization in hormozgan province (Iran). Fresh Produce 5:69-76.