Morphological properties of squash genotypes
In this study, 14 squash genotypes belonging to Cucurbita maxima L. were collected from different locations in Erzincan province. This squash population has been characterized according to morphological and molecular traits. Since changes in morphological traits occurred in response to external conditions, it is important to support these morphological variations with molecular studies. Morphological features of genotypes are given in Tables 3, 4 and 5. It was observed that there were significant morphological differences in plant phenotype, leaf, flower and fruit characteristics among the collected Cucurbita maxima genotypes. The plant growth habit was considered as trailing in 11 genotypes and semi- trailing in 3 genotypes. Branching was determined in all genotypes. Genotypes were divided into 3 groups as weak (3 genotype), medium (4 genotype) and strong (7 genotype) according to degree of branching. In addition, squash genotypes showed variation in terms of leaf characteristics such as leaf attitude of petiole, leaf blade size, incisions of leaf blade and intensity of leaf blade. Leaf attitude of petiole was identified as vertical in 11 genotypes and semi-vertical in 3 genotypes. Leaf blade size was large in 11 genotypes and medium in 3 genotype. Incisions was considered as weak in 3 genotypes and medium in 3 genotypes, whereas in 8 genotypes incisions of leaf blade were absent (Table 3). As with other morphological features, it was observed that there was variation among genotypes in terms of flowers (male and female). It was determined that approximately 4 of the genotypes had ring at inner side of petal and that there was no inner circle in the female flowers of 10 genotypes. Based on the expression of inner circle color grade at petal of male flowers, genotypes are divided into 3 groups as absent, slight and medium. It was observed that 50% of genotypes (7 genotypes) did not have circle at inner side of petal. Genotypes were divided into 2 groups as yellow and orange according to color of pistil of male flower. It was determined that 8 genotypes had yellow and 6 genotypes had orange color. Hairiness on the flower stalk was considered as as weak in 9 genotypes, medium in 3 genotypes and strong in 2 genotypes. Differences were determined between genotypes according to the length of the flower stalk. Genotypes were divided into 3 groups based on these properties. Only 1 genotype were classified as short, 10 genotypes as medium and 3 genotypes as strong (Table 4). In addition, squash genotypes showed high variation in fruit shapes and skin colours. It was determined that fruit shape of 9 genotypes were transverse wide elliptical, 3 genotypes were wide elliptical, 1 genotype were elliptical and 1 genotype was napiform. Six different colors were determined as the major colour of skins of the squash genotypes: whitish (8 genotypes), grey (1 genotypes), grey-green (2 genotypes), grey-green-orange (1 genotypes), orange-green (1 genotypes) and cream-green (1 genotypes) (Table 5).
Table 3
Plant and leaf morphological parameters of squash genotypes
Genotypes | Plant | | Leaf |
Growth habit | Branching | Degree of branching | | Leaf attitude of petiole | Leaf blade size | Incisions | Intensity of blade green color |
≠ 5 | Trailing | Present | Strong | | Vertical | Large | Absent | Medium |
≠ 12 | Trailing | Present | Strong | | Vertical | Medium | Absent | Medium |
≠ 15 | Semi trailing | Present | Weak | | Semi vertical | Large | Weak | Dark |
≠ 16 | Trailing | Present | Weak | | Semi vertical | Large | Weak | Dark |
≠ 18 | Trailing | Present | Strong | | Vertical | Large | Absent | Dark |
≠ 20 | Trailing | Present | Medium | | Vertical | Large | Medium | Dark |
≠ 21 | Trailing | Present | Medium | | Vertical | Large | Weak | Dark |
≠ 22 | Trailing | Present | Medium | | Vertical | Large | Medium | Dark |
≠ 24 | Trailing | Present | Strong | | Semi vertical | Large | Absent | Dark |
≠ 43 | Trailing | Present | Strong | | Vertical | Medium | Absent | Medium |
≠ 44 | Trailing | Present | Strong | | Vertical | Large | Absent | Medium |
≠ 45 | Trailing | Present | Strong | | Vertical | Large | Absent | Medium |
≠ 47 | Semi trailing | Present | Weak | | Vertical | Medium | Medium | Medium |
≠ 48 | Semi trailing | Present | Medium | | Vertical | Medium | Very strong | Medium |
Table 4
Flower morphological parameters of squash genotypes
Genotypes | Female flower | | Male flower |
Inner side of petal | Pistil color | | Petal inner circle color grade | Sepal leaf length | The length of the flower stalk | Hairiness on the flower stalk |
≠ 5 | Absent | Yellow | | Slight | Medium | Medium | Weak |
≠ 12 | Absent | Yellow | | Slight | Medium | Medium | Strong |
≠ 15 | Absent | Orange | | Slight | Medium | Long | Weak |
≠ 16 | Absent | Orange | | Absent | Medium | Medium | Medium |
≠ 18 | Absent | Yellow | | Absent | Medium | Medium | Weak |
≠ 20 | Absent | Yellow | | Slight | Medium | Medium | Medium |
≠ 21 | Present | Yellow | | Absent | Medium | Long | Weak |
≠ 22 | Present | Orange | | Absent | Medium | Long | Medium |
≠ 24 | Absent | Orange | | Slight | Medium | Medium | Strong |
≠ 43 | Present | Yellow | | Absent | Medium | Medium | Weak |
≠ 44 | Absent | Yellow | | Absent | Medium | Medium | Weak |
≠ 45 | Absent | Yellow | | Medium | Medium | Medium | Weak |
≠ 47 | Absent | Orange | | Very strong | Medium | Short | Weak |
≠ 48 | Present | Orange | | Absent | Medium | Medium | Weak |
Table 5
Fruit morphological parameters of squash genotypes
Genotypes | Fruit |
Shape | Main color of skin | Intensity of skin main color | Number of skin color | Diameter | Length |
≠ 5 | Transverse wide elliptical | Whitish | Light | One | Large | Long |
≠ 12 | Transverse wide elliptical | Whitish | Light | One | Large | Long |
≠ 15 | Elliptical | Grey | Light | One | Large | Long |
≠ 16 | Transverse wide elliptical | Grey green | Dark | One | Large | Medium |
≠ 18 | Transverse wide elliptical | Whitish | Light | One | Large | Long |
≠ 20 | Wide elliptical | Whitish | Light | One | Large | Long |
≠ 21 | Transverse wide elliptical | Whitish | Light | One | Large | Long |
≠ 22 | Transverse wide elliptical | Whitish | Light | One | Large | Medium |
≠ 24 | Wide elliptical | Whitish | Light | One | Large | Medium |
≠ 43 | Wide elliptical | Grey-green-orange | Light | Two | Large | Medium |
≠ 44 | Transverse wide elliptical | Grey green | Light | One | Large | Long |
≠ 45 | Transverse wide elliptical | Whitish | Light | One | Large | Long |
≠ 47 | Transverse wide elliptical | Orange-green | Medium | One | Medium | Short |
≠ 48 | Napiform | Cream-green | Light | Two | Small | Short |
SSR Analysis
The 7 SSR markers used in our study produced a total of 23 polymorphic bands, the number of alleles per marker ranged from 2 (GMT-M30 marker) to 5 (GMT-P68 marker) and the mean number of alleles was 3.286 (Table 6). Polymorphic information content (PIC) is an important value that evaluates the efficiency of polymorphic loci and determines the discrimination ability of markers. The PIC value ranges from 0.00 (GMT-M30) to 0.202 (GMT-P68), with a mean of 0.13. The markers GMT-P25 and GMT-P68 were found to be the best among the markers used to discriminate between genotypes due to their higher PIC values (Table 6). Gene diversity ranges from 0.064 (GMT-M61) to 0.314 (GMT-P41), with a mean of 0.181.
Table 6
Allele number, polymorphic allele number, polymorphism percentage and PIC values of iBPS markers
Number | Primer | Number of Alleles | Major Allele Frequency | Gene Diversity | PIC* | He** |
1 | GMT-P41 | 3,000 | 0,857 | 0,314 | 0,162 | 0.143 |
2 | GMT-M61 | 3,000 | 0,893 | 0,064 | 0,140 | 0.107 |
3 | GMT-P68 | 5,000 | 0,833 | 0,212 | 0,190 | 0.214 |
4 | GMT-M259 | 3,000 | 0,929 | 0,119 | 0,107 | 0.071 |
5 | GMT-P18 | 3,000 | 0,929 | 0,183 | 0,107 | 0.036 |
6 | GMT-P25 | 4,000 | 0,857 | 0,283 | 0,202 | 0.036 |
7 | GMT-M30 | 2,000 | 1,000 | 0,093 | 0,000 | 0,000 |
Mean | 3,286 | 0,900 | 0.181 | 0.130 | 0.087 |
Total | 23 | | | | |
*PIC- Polymorphic information content, **He (expected heterozygosity) |
Cluster analyzes and principal component analyzes for SSR markers
Comparative analysis of molecular sequence data enables the determination of proximity or distance between genotypes as well as the construction of a phylogenetic tree for clustering genotypes. For this purpose, cluster analysis was performed between squash genotypes using UPGMA based on Nei's genetic distance. According to the results of this analysis, four major clusters were formed. Dice genetic similarity coefficient was used to estimate genetic diversity. This coefficient is often used to estimate genetic distance. The highest genetic difference (0.63) was found between genotypes ≠ 36 and ≠ 46 genotypes. As a result of the analysis, squash genotypes were divided into three major groups. In the second group, only single genotype of Çayırlı location (≠ 18, ≠ 20) was determined. In the first cluster, two genotype was found Cevizli (≠ 47, ≠ 48) locations. In the third group, mostly genotypes of Bahçeliköy (≠ 5), Cevizli (≠ 43, ≠ 44, ≠45), Çayırlı (≠ 15, ≠ 16≠, ≠ 21, ≠22, ≠ 24) and Çatalarmut (≠ 12) locations were included. (Fig. 1).
Principle component analysis (PCoA) presents spatial distribution of relative genetic distance between the populations [30]. In present study, PCA analysis was performed for better and more detailed visualization of the variation within and between the populations. With the aid this method, a 2-D diagram is generated based on closeness or distance matrix between the genotypes and the distances between the resultant groups put forth the actual distances (Mohammadi and Prasanna, 2003). According to present findings, the genotypes Çayırlı (≠ 22) and Cevizli (≠ 47, ≠ 48) were placed on upper left section of the Principle Axis-1. The genotypes Çayırlı (≠ 18, ≠ 20) and Cevizli (≠ 44) were gathered on lower left section of Axis-1. The genotypes Çatalarmut (≠ 6), Çayırlı (≠ 16, ≠ 21, ≠24) and Cevizli (≠ 43, ≠ 45) were placed on lower right section of Axis − 1. The genotypes Bahçeliköy (≠ 5) and Çayırlı (≠ 15) were gathered on upper right section of Axis-1 (Fig. 2).
Genetic Structure Analysis Of Ssr Markers
ΔK is used to determine optimal values of K. The highest value in our study was obtained as K = 4 (Fig. 3). The low population size (K value) in our study is thought to be due to the high gene flow between the sample collection regions. In our study, 2 genotypes were found in the first subpopulation, 2 genotype in the second subpopulation and 10 genotypes in the third subpopulation (Fig. 4; Table 7). The FST (F-statistics) values in the first, second and third subpopulations were determined as 0.4581, 0.5789 and 0.6112, respectively (Table 8).
Table 7
Membership coefficient of squash genotypes in four subpopulations
Genotype | Sub-population |
1 | 2 | 3 |
G5 | 0.012 | 0.376 | 0.611 |
G12 | 0.007 | 0.382 | 0.610 |
G15 | 0.014 | 0.375 | 0.611 |
G16 | 0.007 | 0.383 | 0.610 |
G18 | 0.008 | 0.595 | 0.397 |
G20 | 0.017 | 0.605 | 0.379 |
G21 | 0.007 | 0.383 | 0.611 |
G22 | 0.059 | 0.441 | 0.500 |
G24 | 0.007 | 0.382 | 0.611 |
G43 | 0.007 | 0.384 | 0.610 |
G44 | 0.025 | 0.527 | 0.447 |
G45 | 0.103 | 0.359 | 0.538 |
G47 | 0.987 | 0.007 | 0.006 |
G48 | 0.964 | 0.020 | 0.016 |
Table 8
Expected heterozygosity and FST values in four squash subpopulations
Sub-population (K) | Expected heterozygosity | FST |
1 | 0,1610 | 0,4581 |
2 | 0,1331 | 0,5789 |
3 | 0,1167 | 0,6112 |
Mean | 0,1369 | 0,5494 |