Document 1:

Abstract

Genetic diversity analysis was undertaken in 42 accessions of geographically distant genotypes (14 from north-east India and

28 from north India) of bottle gourd (Lagenaria siceraria) from India using inter-simple sequence repeat (ISSR) markers. A

total of 209 amplified bands were obtained from 20 ISSR primers used in this study, of which 186 were polymorphic with 89.00

% band polymorphism. Various parameters namely, observed number of alleles, effective number of alleles, Neis gene

diversity/heterozygosity, resolving power, Shanons information index and gene flow were estimated under experiment.

Jaccards similarity coefficient matrix was generated for pairwise comparisons between individual ISSR profiles and UPGMA

cluster analysis based on this matrix showed clustering into six groups. Jaccards coefficient of similarity values (GS) ranged

from 0.409 to 0.847, with a mean of 0.628 revealing a moderate level of genetic diversity (GD). The Bayesian model-based

approach to infer hidden genetic population structures using the multilocus ISSR markers revealed two populations among

the 42 genotypes. This is the first report on the assessment of genetic variation by using ISSR markers in this medicinal plant

and this study of diversity analysis will be helpful in analysing future hybrid breeding strategy and devising effective

germplasm exploration and conservation strategy.

Introduction

Twenty primers that produced clear, distinct and reproducible patterns across all the 42 accessions were used for band scoring and

genetic analysis. They generated a total of total 209 loci (bands) ranging from 150bp to 1500bp. Over all, the level of Percent Band

Polymorphism (PBP) detected by these 20 ISSR primers ranged from 75 to 100 (Table 2). Average percent band polymorphism (PBP)

detected was 88.49 % polymorphism with 186 polymorphic loci in the bottle gourd germplasm. Maximum band polymorphism for

north India is 85.17 %, and north-east India is 75.60%. Resolving power (Rp) measures the ability of primers to distinguish between

genotypes (Prevost & Wilkinson. 1999 and is based on the distribution of detected bands within the sampled population. Primer

sequence CAC ACA CAC ACA CAC ARC G, showed the maximum RP value of 8.105 and (GA)8 T shows least Rp value 1.421,

indicating their resolving capacity in population. The primers with high Rp ((GA) 9 AT and C(AC)7 ARC G) can be used for varietal

discrimination and DNA fingerprinting studies. A further sequence investigation and conversion of these ISSR markers into sequence

characterized amplified region (SCAR) markers would be very useful for a fast and effective identification of its medicinally

importance genes.

Inter-population analysis between north Indian and north-east Indian population revealed that observed numbers of alleles and
expected no. of alleles were 1.8900 and 1.5385 respectively, shown in Table 3.
 Neis Gene Diversity which is equivalent to the average heterozygosity in the above study was 0.3144

0.1658. Another genetic diversity parameter which was obtained was Shanons information index with value of

0.4696 0.2227. These results indicate that populations harbor moderate inter population diversity, thereby,

enhancing their chances to survive under variable climatic conditions. High gene flow value was obtained (12.99)

for the above two populations. This gene flow value suggests a high degree of movement of the germplasm in the

two areas and may also be due to the cross-pollinated nature of the crop. The Neis genetic distance between the

populations of north India and north east India was 0.0203 which indicates that both north-east Indian population and

north Indian population are genetically similar to each other. There is a high rate of movement of germplasm within

India that may be responsible for the low genetic distance between the two populations.

The binary scoring data of the 20 primers was subjected to NTSYS-pc 2.01 software for deriving the UPGMA

dendrogram based on the Jaccards coefficient for similarity indexing and is presented in Figure 1. UPGMA based

dendrogram does not show correlation with the geographical locations. The obtained matrix showed that Jaccards

coefficient of similarity values (GS) ranging from 0.409 to 0.847, with a mean of 0.628 revealing a moderate level

of genetic diversity (GD) within these 42 germplasm. The smallest similarity value (0.409) suggested the high

divergence between Pusa Sandesh and BG49 and the maximum similarity value (0.847) was scored between BG45

and BG46 indicating that both cultivars were the most similar. The dendrogram for 42 bottle gourd germplasm

revealed two main clusters; Cluster I was the largest, comprising 38 germplasms which can be divided into numbers

of sub clusters, and cluster II is very small consist of 3 germplasm only. In cluster I there are two cultivars which

were present as out group, that are BG13 and BG30. BG49 is present as an out group outside of these clusters.

Bottle gourd is a cross pollinated crop, allowing admixture between different genotypes to occur. Based on

STRUCTURE analysis, the value of K peaked at K=2 (Fig. 3), hence the number of populations detected in the

accessions were 2. Overall proportion of membership of the samples in each of the 2 populations were 0.527 and

0.413. To ration the grade of mingling, we executed population structure analysis and the results specify that all the

samples have a high genetic relationship in their specific cluster (>60%) with few accessions (BG 40, BG 56, BG

21, BG 17, BG 16, BG 23 and BG 26) showing the contribution of the other population in their ancestry. This result

argues that there has been a lot of gene flow in these accessions as was also revealed by the high gene flow value

above. Also there were no geographic trends visible in the population structure.
In our study, ISSR markers proved to be useful tool for identifying individuals and assessing the genetic structure in

L.scieraria germplasm. This is the first report on the assessment of genetic variation by using ISSR markers in this

medicinal plant. The present data provide enough evidence of its high applicability in diversity analysis of

L.scieraria crop.