Characterisation of pumpkin (Cucurbita moschata duch.) varieties through morphological and molecular markers

Abstract

Pumpkin (Cucurbita moschata Duch.), a crop of Central Mexican origin belonging to the family Cucurbitaceae, is popularly cultivated and valued in Kerala as a vegetable. The tender, large and often round immature fruits of pumpkin with a thick, smooth to slightly ribbed skin, which is mostly deep yellow to orange in colour, is an integral part of the Kerala cuisine. Despite its popularity in the state, few high yielding varieties are in cultivation. To ensure increased production, availability of high quality seeds of improved varieties or hybrids has to be guaranteed. Pumpkin being a cross pollinated crop, occurrence of cross contamination during its seed programme cannot be overruled. Hence, ensuring the purity and identity of seeds of the variety before sale becomes inevitable.

Considering the importance of varietal identification in maintaining the genuineness and quality of seeds in seed production programmes, the present investigation envisaged to characterise six pumpkin varieties in the seed chain using morphological and molecular markers, and to generate fingerprints or molecular ID’s of the six varieties using selected polymorphic Inter-simple sequence repeats (ISSR) and Simple sequence repeats (SSR) markers.

Characterisation of pumpkin varieties based on 28 quantitative and 16 qualitative traits was done using DUS and NBPGR descriptors. Qualitative vegetative traits like tendril characteristics (presence or absence of tendril, nature of coiling and branching) and leaf shape were not useful for grouping the varieties. Similarly, among the qualitative fruit characteristics, waxiness of mature fruit skin also proved insufficient to distinguish the varieties.

Fruit shape was round flat in varieties Ambili, Suvarna and CO-2, while it was elongate/oblong in Saras, club shaped in CO-1 and flattish round in Arka Chandan. Based on qualitative traits, variety Arka Chandan could be clearly distinguished from the other varieties based on poor early growth vigour, moderately incised leaf blade margin, absence of silver patches on leaf blade, flattish round fruit shape, light green immature fruits and dark orange fruit flesh colour. In addition, the seeds of the Arka Chandan had a characteristic marking on the dorso-ventral surfaces unlike other varieties. Quantitative traits proved to be more useful than the qualitative traits for effective identification and categorisation of varieties. Results revealed that among the quantitative traits studied, leaf dimensions (blade length and width) and length of petiole could not be employed for distinguishing the six varieties. Variety Ambili flowered the earliest (49.25 days) and also possessed highly pubescent leaves, while variety Arka Chandan was late flowering (68.00 days). Peduncle length, fruit length and most of the seed dimensions (seed count per fruit, 100 seed weight, width and thickness of seed) was the least in this variety. In general, the size of seeds in varieties CO-1 and CO-2 was higher than those of others. Cluster analysis grouped Arka Chandan (Cluster V) and varieties CO-1 and CO-2 (Cluster IV) the farthest with an inter-cluster distance of 212.25. Principal component (PC) analysis indicated that trait components in PC1 registered an Eigen value of 16.79 and the traits in PC1 contributed 58 per cent to the variability among the varieties, emphasising their utility in identification of varieties. Among the 33 ISSR markers, 28 exhibited polymorphism. The total number of amplicons detected by an individual primer ranged from 4 in UBC-818 to 18 in UBC-847. High polymorphic information content (PIC) value was observed in UBC-809, whereas, low PIC was recorded in UBC-818. The six varieties grouped into four clusters based on ISSR binary data. Out of 20 SSR markers used for genotyping, only five showed polymorphism. The highest Jaccard’s similarity value (1.00) was observed between Saras and Suvarna. The most dissimilar varieties were Saras and Arka Chandan, and Suvarna and Arka Chandan, with a similarity coefficient of 0.12 each. The clustering algorithm grouped the varieties into four clusters.

The polymorphic SSRs could be efficiently utilised for distinguishing Arka Chandan and therefore can prove useful for testing the genetic purity of this variety. Unique bands producing ISSR markers were used to generate variety specific DNA fingerprints. No single primer per se proved useful in distinguishing all six pumpkin varieties. However, ISSR primer UBC-822 could distinguish four out of six varieties studied. It produced unique amplicons of size 473 bp, 552 bp, 1403 bp and 517 bp, respectively in Ambili, Saras, CO-1 and Arka Chandan, proving its utility in testing for genuiness and purity of seed lot. In general, it can be concluded that the correlation that existed between morphological and molecular assessments was of medium magnitude. The absence of high consensus between the assessments should not be considered a limitation of these tools to characterize and quantify variability. It only indicates that both morphological and molecular characterisation is important and play a complementary role in providing a better understanding and differentiation of the pumpkin varieties.