Assessment of the genetic variability amongst mandarin (Citrus reticulata Blanco) accessions in Bhutan using AFLP markers

BACKGROUND: Bhutan is a small Himalayan country lying within the region considered to be the origin of citrus. Diverse citrus wild types grow naturally in different climates, elevations and edaphic conditions, but only mandarin is cultivated commercially. The first report of Huanglongbing (also known as greening disease) in Bhutan in 2003, and the threat it posed to the country’s citrus orchards prompted the collection of mandarin germplasm from across the country. This paper describes the genetic diversity of mandarin accessions in Bhutan using amplified fragment length polymorphic (AFLP) markers. RESULTS: Twenty three accessions of Bhutanese mandarin were analyzed using AFLP markers to assess the genetic variability that is believed to exist only in Bhutan and some parts of North East India and South China. Five primer pairs (E-ACA/M-CAG, E-ACG/M-CAT, E-ACC/M-CTT, E-AAG/M-CAA and E-ACA/M-CTC) were identified (based on the number and quality of polymorphic bands produced) and used for the analyses. A total of 244 bands were scored visually of which 126 (52%) were polymorphic with an average polymorphism information content of 0.95 per marker. A cluster dendrogram based on multiscale bootstrap sampling categorized twenty three accessions into two broad groups containing eight and 14 accessions, respectively. Group A consisted accessions (Tsirang1, Tsirang3, Sarpang1, Dagana4, Samtse4, Dagana1, and Trongsa2) from five districts (Tsirang, Sarpang, Samtse, Dagana and Trongsa) and their grouping was strongly supported by bootstrap analysis (B p-value = 96%, AU p-value = 86%). Cluster B consisted of 14 accessions divided into three sub-groups (1, 2 and 3). However, bootstrap value supported significantly for subgroup1 (containing accessions: Tsirang4, Sarpang5, and Tsirang2) and subgroup3 (with accessions - Zhemgang2, Zhemgang3 and Zhemgang4). CONCLUSION: This study indicates that Bhutanese mandarin germplasm collected from across the country are genetically diverse although the level of variability differed among the accessions assessed. The variation in genetic variability was observed irrespective of where the accessions were collected suggesting that phenotype and geographical location can serve a basis for future germplasm collection in Bhutan. Further, five primer pair combinations could separate 23 mandarins accessions considered in this study, suggesting that AFLP markers can be a useful tool for future identification.