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Genetic diversity of elite wheat mutant lines using morphological characters and molecular markers

Genetic diversity is the material basis for crop improvement. The genetic diversity of 17 wheat genotypes was evaluated using 25 agro-morphological characters and 10 simple sequence repeat (SSR) markers. The objective of this study was to determine the genetic diversity of elite stem rust resistant mutant lines in comparison with their adaptable but susceptible parent varieties using morphological traits and molecular markers. The results obtained showed significant variation in morphological traits and molecular makers existed. Morphological diversity between mutant lines and their parent varieties was mainly separated by grain yield per spike, 1000 grain weight and maturity time period. The dendrogram based on 10 SSR markers grouped the 17 genotypes into three major clusters and six sub-clusters with mutants clustering with their respective parents. 10 SSR primer pairs yielded 13 polymorphic loci with a percentage of 92.86%. The mean number of alleles per locus in each group was 2.0 and the mean number of polymorphic alleles per locus was 1.9286. The gene diversity ranged from 0 to 0.4893 for each sample. Results showed it is possible to classify genetic diversity of elite wheat genotypes and select them for the highest genetic diversity. The results can be used in selecting diverse parents in breeding programs and also in maintaining genetic variation in the germplasm. addition, Hardy-Weinberg equilibrium (HWE) was tested by the Chi-squared test. Gene diversity (GD) and polymorphic information content (PIC) were measured by calculating the shared allele frequencies (Weir, 1996) using PowerMarker 3.25 (Liu and Muse, 2005). UPGMA algorithm was used to construct an unrooted phylogram from a distance matrix based on (Nei, 1973) genetic distances, using MEGA4 software implemented in PowerMarker 3.25 (Liu and Muse, 2005).


INTRODUCTION
Wheat (Triticum aestivum.L) contributes to food security in Kenya and his ranked second important cereal crop after maize (KALRO, 2016). However, its productivity is low due to abiotic and biotic stresses (Njau et al., 2010). Wheat is a self-pollinating crop that has been bred and developed for specific end-use quality traits and to grow within a specific production environment. Genetic variability holds the potential to deal with multiple biotic and abiotic stresses. Knowledge of genetic diversity of a crop is important in the development and improvement of a particular crop species. Evaluation of genetic diversity among adapted germplasm provides predictive estimates of genetic variations among segregating progeny for new varieties development. It is desirable therefore to have a large genetic diversity for the creation of new genotypes.
Morphological traits and molecular markers play an important role in the analysis of variance in genetic diversity studies. Use of morphological traits alone is unreliable because they are greatly influenced by the environment (Takumi et al., 2009). A genotype may exhibit different morphological traits for two different locations (Stepien et al., 2007). Molecular markers increase the breeding progress for traits that are difficult to select under field conditions and those that are controlled by multiple genes. Simple Sequence Repeats (SSRs) were considered the best molecular markers because they are able to identify and differentiate genotypes within a particular species. Their co-dominant inheritance and the high level of polymorphism in a large sample of elite germplasm make them useful (Prasad et al., 2000). Genetic diversity is important for adaptability and survival of genotypes against the threats of biotic and abiotic stresses. Wheat breeding in Kenya has attempted to develop resistant wheat varieties through utilization of resistant genes, but virulence is still being reported in most these varieties (Njau et al., 2009). The objective of this study was to assess genetic diversity of eight pre-selected elite mutant lines in comparison with their adaptive but stem rust susceptible parents and seven other commercial checks using morphological traits and molecular markers.

Experimental local sites
Three major wheat growing sites were used in Kenya; the first site was at University of Eldoret, on 0°34ʹN; 35° 18 ʹE, at 2,153 m above sea level. The average temperature is 18°C with average annual rainfall of 1,100 mm. Second site was at KALRO-Kitale, on 0°33ʹS; 35° 55ʹE, at 2,900 m above sea level with average temperatures of 15°C and average rainfall of 1,800 mm. Third site was KALRO-Njoro, on 0°20ʹS; 35° 56ʹE, at 2,185 m above sea level with average temperatures of 20°C and average annual rainfall of 900 mm. The experiments were carried out in 2012/2013.

Plant materials
Seventeen wheat genotypes were used in this study comprising of eight pre-selected mutant lines as illustrated in Table 1, from University of Eldoret: SP-9, SP-16, SP-20, SP-21, SP-26, SP-29, SP-31 and SP-34. Two parental varieties: Njoro II (SP-N) and Kwale (SP-K) and seven other commercial checks: Duma (SP-D), Pasa (SP-P), Simba (SP-S), Farasi (SP-F), Robin (SP-R), KS Mwamba (SP-M) and Chozi (SP-C) all sourced from KALRO Njoro Seed Unit. The two parents and the seven commercial check varieties used in this study are popular and moderately susceptible commercial wheat varieties grown in Kenya.

Field experimental procedures
The 17 genotypes were established based on Complete Randomized Block Design with three replications per location. Field experimental plots were 6 rows by 2 m in length with 20 cm inter-row by 5 cm intra-row spacing. Seeds were hand planted with Di-ammonium Phosphate (DAP 18:46:0) at a rate of 125 kg/ha, followed by an application of Urea at 75 kg/ha at tillering and booting stages. Irrigation was carried out when the soils were dried to maintain soil moisture. Wheat agronomic practices were carried out as recommended by Kinyua and Ochieng (2005).

Green house experimental procedure
Seeds (10) of each genotype were planted in the greenhouse in pots using completely randomized design (CRD).

Data collected for morphological analysis
Plants were selected at random for 25 morphological characters and were evaluated to determine morphological diversity. They were divided into qualitative and quantitative traits and were measured or observed as follows: germination (%), plant height (cm), spike length (cm) and flag leaf area (cm 2 ). Growth habit, seed shape, re-curved flag leaf, spike shape, flag leaf attitude, straw pith, spike density, grain colour, lower glume, sprouting, awns, and shriveling were observed. Number of tillers (no.), lodging (no.), number of grains per spikelet (no.), number of spikelet's per spike (no.), awn length (cm), seed diameter (cm), 50% heading (Days), 50% maturity period (Days), grain yield per spike (g), and 1000 seed weight (g).

Statistical analyses
Analyses of variance (ANOVA) were performed on the quantitative traits using Genstat computer software (Gensat 15th Edition, 2012). Data were subjected to general linear model and analyzed as a RCBD: Y ijkl = µ + λ i + π (j)i + t k + λt jk + Ɛ ijkl where Y ijkl = plot observations, µ = overall mean of experiment, λ i = Season effect, π (j)i = Replication within season effect, t k = genotype effect, λt jk = Interaction of genotype effect, Ɛ ijkl = Residual effect.
Qualitative data were subjected to frequency distribution analyses and assigned numerical values and computed in excel using Shannon-Weavers diversity index (H') = (log e Pi) / log e n; where H' = Shannons-weaver diversity index, Pi = Frequency proportion of each qualitative trait, n = number of classes per qualitative trait; H' value ranges from 0-1 (0 = absences of diversity and 1 maximum diversity). Table 2 were used to investigate the relationship among the 17 genotypes. 10 seeds of each genotype were planted in pots in the greenhouse and after 4 weeks, leaf tissues were selected randomly from each genotype, cut and crushed together in the laboratory using a mortar and pestle. DNA extraction was performed following modified cetyltrimethylammonium bromide (CTAB) protocol (Doyle, 1990). 45 μl of SDS was added and mixed thoroughly. Samples were then incubated at 65°C in a water bath for 1 h and cooled down for 5 min before adding 220 μl of 5M Potassium Acetate. Samples were then put in ice for 15 min and later on centrifuged for 10 min at 13000 rpm. 700 μl of the supernatant was transferred into a microfuge tube and 600 μl of Chloroform and Isoamyl Alcohol in the ratio (24:1) added and centrifuged again for 10 min at 13000 rpm. 600 μl of the supernatant was transferred into a new tube and ice cold isopropanol added to the samples. They were centrifuged again for 10 min at 13000 rpm to pellet the DNA. The supernatant was poured leaving the pellet. 500 μl of 70% ethanol was added to the DNA pellets and centrifuged at 6500 rpm for 5 min and then gently poured off. The pellets were air dried for 1 h and resuspended in50 μl distilled water then stored at 4°C. Concentration and purity of the extracted DNA was determined using Nanodrop200 spectrophotometer (Thermo Fisher Scientific Inc.) and Gel electrophoresis. All samples exhibited good quality and quantity of DNA for PCR amplification. 1 g of agarose was added to 100 ml of TBE buffer (Tris Boric Edta) and casted to make the gel that was used to quantify the DNA samples (1% gel). Nanodrop spectrophotometer 200 (Applied Biosystems) was used to quantify the extracts. The extracted total nucleic acid was suspended in distilled water and 1 ul of each sample loaded on the spectrophotometer pedant and its absorbance measured. Extracts were run in a 1% agarose gel containing Ethidium bromide staining dye at voltage of 100 V and a current of 400 mA for 30 min and visualized on a UV Trans illuminator. The study used a scoring method where (1) represented presence of expected band, while (0) was absence of band. Genetic variation at each locus was characterized in terms of observed number of alleles (na), observed heterozygosity (HO), expected heterozygosity (HE), gene diversity and Shannon's diversity index (I) using the genetic analysis packages POPGENE Version 1.32 (Yeh et al., 2000).

SSR markers as seen in
In addition, Hardy-Weinberg equilibrium (HWE) was tested by the Chi-squared test. Gene diversity (GD) and polymorphic information content (PIC) were measured by calculating the shared allele frequencies (Weir, 1996) using PowerMarker 3.25 (Liu and Muse, 2005). UPGMA algorithm was used to construct an unrooted phylogram from a distance matrix based on (Nei, 1973) genetic distances, using MEGA4 software implemented in PowerMarker 3.25 (Liu and Muse, 2005).

Qualitative traits
Results of qualitative traits showed 94% of the genotypes had erect growth habit, 34% had shriveled grains, 24% had hard red grains, while 36% were soft white grains. Computed diversity ranged from 0.27 to 0.85. Low diversity values for straw pith (0.27) indicated a low variation while shriveled grains showed high variation (0.85) among the 17 genotypes (Table 3).

Quantitative traits
The combined analysis of variance (ANOVA) showed the 17 genotypes were significantly different (P < 0.05) for seed weight, seed diameter, spike length, grain yield per spike and number of grains per spike. The seasonal effects were significant (P < 0.05) for seed weight, seed diameter, days to maturity, spike length, plant height and grain yield per spike. But no significant difference (P < 0.05) was observed from awn length and days to 50% ear emergence. Significant (P < 0.001) genotype × season (G × S) interaction were observed for seed weight, seed diameter, spike length, grain yield per spike and number of grains per spike (Table 4).

Pearson's moment correlation
Pearson's correlation (r) showed significant positive correlation between seed weight and seed diameter, seed weight and grain yield per spike. However, significant negative correlation was between seed weight and number of tillers, seed weight and maturity period (Table 6).

Genetic diversity of 17 wheat genotypes based on SSR markers
Total of 10 polymorphic SSR primers were detected after  screening 20 markers on 17 genotypes. Most primers had 2 alleles and the alleles sizes were within the expected range. The 10 SSR primer pairs yielded a total 13 polymorphic loci with a percentage of 92.86%. The mean number of different alleles per locus in each group was 2.0 and the mean number of polymorphic alleles per locus was 1.9286 ( Figure 1).
The expected and observed moments of heterozygosity was calculated to estimate the number of heterozygous loci. The expected heterozygosity (HE) and observed heterozygosity (HO) ranged from 121.53 to 1.49 and from 22.75 to 0.642, respectively (Figure 2).
The number of alleles obtained was low compared to other studies (Blair et al., 2010). This finding can be attributed to high genetic similarity between the accessions or crossbreeding between the accessions. The gene frequency varied from 0.8824 for Sr2 allele 2 to as low as 0.05882 for Sr21 allele 3 (Figure 3).

Cluster analysis
Unrooted phylogenetic tree was constructed using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) agglomerative hierarchical clustering (Figure    5). The dendrogram generated from the results showed the evaluated wheat genotypes segregate into three major clusters and six sub-clusters. The mutants clustered with their respective parental varieties as their resistance profiles were similar or related significantly all the mutants segregated with their parental varieties and hence resistance profiles of parents can be used as references to characterize resistance of the mutant lines to stem rust.

DISCUSSION
Morphological diversity existed between mutants, their parents and other commercial checks used in this study. Both qualitative and quantitative traits showed diversity. This was supported by the average Shannons-Weavers index for qualitative traits (Table 3). The genotype × season (G × S) interaction observed on number of grains per spike, maturity time period, number of tillers  and 1000 seed weight showed the influence of seasonal differences (Table 4). With the necessity for early maturing varieties, there exists a correlation between growth habits, heading time and maturity time period with most genotypes having erect growth habit. Two types of grain textures the soft white grains and the hard red grains were exhibited by the genotypes. The hard red grains are most preferred by bakers and farmers and weighed from 35 to 45 g while the soft white grains weighed 30 to 35 g/1000 grains weight with a moisture content of 13.5%. Spike traits and number of tillers per plant were major traits that separated the mutants from their parent varieties (Table 5). The number of tillers and number of grains per spike was greatly influenced by the nutrients supplied and environmental conditions. Differences in grain texture and spike traits contributed significantly to variability between the mutants and their parent. The mutants and their parents clustered into three major clusters with mutants clustering with their respective parents an indication of closer relationships. Positive correlations were observed between seed weight and grain yields per spike. Seed weight affected grain yield per spike which influence the final yield. Correlation between number of grains per spike, seed weight and grain yield per spike have been recorded by Leilah and Al-Khateeb (2005), who observed a negative correlation between number of grains per spike and seed weight as more grains per spike would tend to reduce the size of grains. Negative correlation was observed between spike length and number of tillers per plant and this was attributed to reduction in food to cater for grains per spike. Negative correlation was also observed between maturity period and grain yield per spike. Genotypes with longer maturity periods had reduced seed weight due to unreliable weather conditions. The polymorphic SSRs markers used were usefully in producing informative bands (Plate 1). Most of the SSR used were polymorphic across the 17 genotypes and a total of 13 alleles were detected with an average number of 2 alleles per locus. According to Salem et al. (2008), the number of alleles per locus ranged from 2 alleles to 7 alleles with an average of 3.2 alleles per locus while Jain et al. (2004) also reported that the number allele per locus ranged from 3 to as high as 22 with an average of 7.8 alleles per locus. Gene diversity ranged from 0 to 0.4893 for each sample, with an average of 0.3361 (Figure 3). The genetic distance analysis separated the 17 genotypes into 3 major clusters and 6 sub-clusters. The genotypes belonging to the same sub-cluster were genetically similar while those belonging to the different sub-clusters were different from each other. The SSRs used in this study demonstrate the ability of SSRs to produce unique DNA profiles and establish discrete identity. Wide range of genetic diversity was observed and it is possible to classify the genetic diversity of the elite mutant lines and select mutant lines for the highest genetic diversity. These findings demonstrated the usefulness and efficiency of SSRs markers in analyzing genomic diversity. According to Hayden et al. (2006), genotypes with the most distinct DNA profile contain the greatest number of novel genes and are likely to carry unique and potentially agronomical useful genes. The genetic diversity levels observed is potentially valuable in predicting sources for selection of genetic diversity with an objective of broadening the wheat genetic base and also have increased progeny performance for complex traits such as yield and disease resistance in wheat production.

Conclusion
Considerable amounts of genetic diversity were observed between the mutants, parents and commercial checks varieties. There was low genetic distance between the genotypes in each sub-cluster attributed to the high genetic similarity between the mutants, their parents and the commercial checks. Observed heterozygosity was higher than expected heterozygosity due to the high genetic variations between the genotypes and within the groupings there were high similarities due to the close relationships and the effects of intense selection in search of the good quality attributes. Sr2 was the most polymorphic marker of the ten SSRs as it exhibited greater ability to distinguish between the different genotypes. These results confirmed the relationships between the parents and their respective mutants being placed into the same groupings on the basis of their genetic similarities. Genetic diversity studies is important in developing strategies in wheat breeding as it can be used in selecting genotypes with certain desired traits for breeding programs. The SSRs confirmed morphological traits information about wheat genetic similarities and variations mostly being separated by their grain characteristics. These results can be used in selecting diverse parents in breeding in order to utilize their genetic potential for progeny improvements. This study contributed to stable wheat production by discovering traits relationship that can be used in breeding purposes for adaptation to various desired conditions while the informative SSR markers can be used to map out traits and aid marker assisted selections.