Alfalfa Improvement in Oman – Prospect and Procedures

1.2.3 Alfalfa Growing Regions

The major alfalfa growing regions are North Batinah (3190 ha), South Batinah (2411 ha), Dhahira (1662 ha) and Interior (1009 ha), which together share 84.70% of total alfalfa area. Interestingly, of these major growing regions, South Batinah (20.65%) has highest percentage of its area in alfalfa followed by Dhahira (17.64%), North Batinah (15.45%) and Interior (14.63%).

The planting is usually done between November and January. The crop is cut first after about 60 to 70 days and then every 25-40 days up to 10-11 times in a year. Typical yield of green matter is about 40 t/ha/year. The national average green matter production and productivity of alfalfa from 1990 to 1997 is depicted in Table 2 (MAF, 1990-97).

Table 2. Area, Production and Productivity of Alfalfa from 1990 to 1998 in Oman ¹

Year	Area (ha)	Production          (‘000 t)	Productivity (t/ha)	Productivity (t/ha)/ cut
1990 1991 1992 1993 1994 1995 1996 1997 1998	9000.0 9021.0 9241.0 9240.0 11047.9 11302.5 11344.5 11344.5 11042.9	345.6 360.8 372.7 375.0 445.3 455.6 487.7 490.0 460.5	38.40                    3.84 40.00                    4.00 40.33                    4.03 40.58                    4.06 40.31                    4.03 40.31                    4.03 43.00                    4.30 43.19                    4.32 41.70                    4.17

1. Department of Agriculture Statistics, Directorate General Planning and Projects, Ministry of Agriculture and Fisheries, Sultanate of Oman

1.2.4 Alfafa: A Perennial Legume

From the point of view of the farmers, Alfafa is a perennial legume that is affordable, yet of very high digestibility and nutritive value. This enables the farmers to explore it at affordable prices and be able to reap optimal yields ultimately. It is persistent in nature and upon harvesting, it’s quite voluminous. The fact that weather conditions have minimal effects on its productivity enables it to be transferred to different regions with ease. It’s well known for its idyllic persistence and adaptability in new regions.

Among the number of agro-ecotypes of Oman, perennial locals viz. Oman Interior local and Batinah local are popular as they are stand persistent owing to their adaptability to the normal practice of ground level cutting by the farmers. A number of landraces that have been differentiated over centuries have been recognized mainly on the basis of longevity. The variants of “Batini” type have an expected life span of 8-10 years in the Batinah but they perform poorly when grown in the mountains.

There are local strains in Hajar Mountains which are known to persist over ten years, but they fail when grown down in the coast, where “Qaryati” is popular. There are also distinct types grown in different regions like “Sharqiya” around Sur and “Omani” in Interior region. The strains in the South have been found distinct from those of North. In 1987-1988 IPGRI, previously IBPGR, collected 87 alfalfa landraces. The collection mission covered most of the area of Oman. Presently, 87 alfalfa accessions were conserved in ICARDA Gene Bank (Guarino, 1987; MAF, 1989). In addition, seven accessions of alfalfa have been conserved in National gene bank of USA

 High Productivity

Figure 1: Productivity of Alfalfa

Recent researches ascertain that Alfalfa can produce high yields as well as high quality forage. This is attributable to the inbreeding practices that have ensured production of high quality species. Relative scientific efforts are devotedly geared towards improving its yield and quality. Depending on the environmental conditions and type of soil, alfalfa yields a significant 50 to 100 tons of forage per hectare. Dry matter in such instances ranges from 12 to 19 tons per hectare. Compared to other forage plants, its productivity is relatively high.

1.2.5 Improvement Objectives for Cultivar Development

The ultimate objective of any cultivar improvement program is the development of germplasm that will enhance production of the crop. Alfalfa use varies from production of green forage (fresh or stored) in intensive forage -animal production systems to a legume in pastures and ranges in extensive forage -animal systems. The goals of alfalfa breeding programs vary considerably, depending on the intended use of the germplasm under development. Nonetheless, there are some traits considered important by most alfalfa breeding programs.

• Yield:

Alfalfa is valued for its ability to produce high yields of high-quality forage. Although alfalfa is used in pastures and ranges, most breeding programs attempt to develop cultivars that will perform well under intensive forage production systems. Most alfalfa breeders feel that a cultivar will not be economically successful unless it is adapted to intensive forage programs.

Genetic increases in alfalfa yield have been about 3% per decade (Elliot et al., 1972; Hill and Kalton, 1976). The total increase in yields obtained by farmers has been greater than this, but part of the increase must be attributed to better management and fertility practices. Separation of genetic from non-genetic increases is difficult because some of the total increase has been the result of cultivars better adapted to intensive management and high fertility.

Several reasons can be proposed for the lower rate of genetic improvement for yield in alfalfa than for the grain crops. Evans (1980) suggested that much of the improvement in seed yield was the result of shunting photosynthetic products to organs or plant tissues of greater economic value. This route has not been available to alfalfa breeders because the entire plant is of economic value.  A second possible reason for the lower rate of progress is that alfalfa is perennial with multiple harvests per growing season. The perennial growth habits of alfalfa dictates that the same plot be observed for several years before selections are made. This increases the time per selection cycle, and under such conditions, an equal gain per cycle of selection would translate to a lower rate of gain for a given time period. A third reason for the lower rate of progress in increasing yield may be that alfalfa is an auto-tetraploid (2n=4x=32). The breeding methods that have been effective with diploid crop species are not as effective when applied to alfalfa.

• Pest Resistance:

Increased levels of pest resistance have been a major success of alfalfa breeding. Many alfalfa breeders work cooperatively with plant pathologists or entomologists, and effective techniques for increasing pest resistance have been developed. Procedures for evaluating resistance have been standardized, and susceptible and resistant lines have been identified for many of the disease and insect pests of alfalfa (Elgin, 1984).

Multiple-pest resistance is a major goal of most alfalfa improvement programs today. The most recent cultivars usually have moderate or higher levels of resistance to bacterial wilt, Fusarium wilt (Fusarium oxysporum Schlecht f.sp. medicaginis (Weimer) Snyd. & Hans.), Anthracnose (Colletotrichum trifolii Bain & Essary), Phtophthora root rot, the pea aphid, and the spotted alfalfa aphid. When vertcillium wilt (Vertcillium albo-atrum Reinke & Berth.) was first discovered in the United States, public and private agencies immediately initiated efforts to incorporate resistance into aphid germplasm. Many of the newer cultivars have moderate or higher levels of resistance to verticillium wilt. Germplasm or cultivars with resistance to a number of other alfalfa pests also have been developed.

Increased pest resistance has been an indirect contributor to increased yields. Spectacular differences in yield can be observed when resistant and susceptible cultivars are grown on a site known to harbor a particular disease or insect pest.

Much of the breeding for pest resistance is done in greenhouse and growth- chamber facilities. Most selections made in the greenhouse and growth-chamber facilities are resistant when tested under field conditions.

Although progress in breeding for multiple-pest resistance in alfalfa has been spectacular, suitable resistance to a number of disease and insect pests has not been found, including fusarium root rot and crown rot [Fusarium solani (Mart.) Appel & Wr. and F. roseum Lk. ex Fr. emend. Snyd. & Hans.), the alfalfa blotch leaf minor (Agromyza frontella (Rondani)], and the clover root curculio [Sitonia hispidula (F)]. A degree of tolerance has been found in some cases, like the alfalfa weevil [Hypera postica (Gyllenhall)] and the potato leaf hopper [Empuasca fabae (Harris)], but the level is not great enough to provide protection in severe infestations or epidemics.

The success in breeding for pest resistance depends on developing methods that permit accurate identification of resistant genotypes. Once this is done, a satisfactory level of resistance to most alfalfa pests often can be obtained in three to five cycles of selection. The inability to find resistance to some diseases or insect pests can very likely be attributed to the lack of a suitable method of identifying resistance.

• Quality:

Alfalfa has a higher feeding value than most forage crops. Some effort is being devoted to greater improvement of alfalfa forage quality. Valid improvement program objectives include increased protein concentration, decreased fiber (increased digestibility), and reduction of bloat potential.

• Protein Concentration:

Alfalfa serves as an important on-farm protein source for ruminant animals. In many farm animal operations, the value of the protein from alfalfa is a major economic justification for growing the crop. Alfalfa was the most efficient species discussed by Heichel (1976) for production of protein. Heritability of protein concentration in alfalfa is relatively high, and progress in breeding for higher concentrations can be expected (Hill and Barnes, 1977; Sumberg et al., 1983). Selection for increased protein concentration often indirectly improves other quality constituents (Cooper, 1973). Near-infra-red reflectance spectroscopy is probably the most economical method for measuring protein concentration in alfalfa forage samples (Shenk et al., 1981).

1.2.6 Constraints in Alfalfa Improvement

Fungal diseases such as crown rot are another factors causing evident yield reduction. In addition, the local cultivars and ecotypes of alfalfa in different regions of the country are at present facing the problems of high temperature, drought and/or salinity. Few researches have been carried out in Sultan Qaboos University and at the regional agricultural research stations. Esechie et al. (2002) investigated the effect of N fertilizer on shoot and root growth in salinity-stressed alfalfa. Esechie and Rodriguez (1999) investigated the effects of salinity in leaf growth of alfalfa. Esechie et al. (1998) studied the effects of salinity on biomass production, nodulation and N₂ fixation in an Omani alfalfa accession “Batini”. Esechie and Rodriguez (1998) compared the distribution of ions in the leaf, stem and roots of alfalfa seedlings irrigated with saline solutions during cool season and warm season in Oman. Esechie (1993) investigated response of alfalfa seed germination to salinity and temperature. The NaCl salinity resulted in substantial reductions in growth, N₂ fixation percentage, and total fixed N₂ in alfalfa and  the effect was more pronounced for the second cuttings than the first ones (Tucker et al,1992).

These factors, together, call for improvement of local cultivars through appropriate breeding programs. The task of crop improvement will be more successful when we have thorough knowledge of their genetic variations. These ecotypes are routinely differentiated using morphological descriptors, and although such descriptions are indeed useful from a breeding perspective, they are inadequate for analysis of population genetic structure. Cultivated alfalfa is autotetraploid (2n = 4x = 32) (McCoy and Bingham 1988), cross-pollinated (allogamous) and seed propagated. The genetic progress is slow in this legume species because of its autotetraploidy and allogamy (Julier et al., 2003). One way to identify the maximally diverse parental genotypes is through an evaluation of genetic diversity using molecular markers. Simple sequence repeat (SSR) or microsatellite markers are codominant, abundant and hyper variable molecular markers from eukaryotic genomes that are being widely used in genetic mapping, phylogenetic studies and marker-assisted selection ((He et al. 2003a). The use of SSR loci as polymorphic DNA markers has expanded considerably over the past decade both in the number of studies and in the number of organisms, primarily due to their facility and power for population genetic analyses (Touil et al., 2008).Currently, the number of available SSR markers is still very limited for use in alfalfa (He et al., 2003b). He et al., (2009) developed 78 genomic SSRs obtained from alfalfa with excellent utility for polymorphic assessment and potential application for phylogenetic and genetic mapping studies of alfalfa. Esechie el at., (2009) evaluated the genetic diversity in Omani alfalfa germplasm and found the existence of variability among 15 Oman alfalfa accessions using the RAPD technique. However, further studies are needed to assess Omani alfalfa in respect of prevailing situation of biotic and abiotic factors especially the salinity.

1.3 Problems Related to a Biotic and Biotic Management Factors

1.3.1 Soil and Water Salinity:

Until mid-seventies, water demand and supply were relatively well balanced. Subsequently, high water demand has led to over pumping and prolonged drought has reduced the extent of recharge. These situations have been progressively deteriorating the quality of both water and soil towards salinity. The affected areas are mostly the farms near the coast, which have abundant but saline water (4-16 dS/m). In the Interior and other regions, however, there is occurrence of dryland salinity where the hydrology of an area has been modified by clearance of vegetation and changed land management practices. Salinity of such water and soil has exceeded the limit tolerable by the economic food or forage crops. Thus, changing situation in both water and soil as mentioned above would expectedly affect the future fodder production in the country since major fodder crops like alfalfa are moderately sensitive (Maas and Hoffman, 1977 and Maas, 1986). This fact assumes much importance because of introduction of sprinkler irrigation. Irrigating alfalfa with water having more than 3 meq/l of Na and Cl by sprinklers during daytime cause severe leaf burn and reduce the crop growth. However, resorting to nighttime irrigation leads to the recovery of crop from the injury (FAO, 1973). Such management to reduce the effect of salinity in already existing cultivars does seem to be practicable when the level of salinity rises higher than the limit tolerable by the crop. Under such circumstances, it would be appropriate and the only approach, to breed genotypes in alfalfa that would be tolerant to desired level of salinity.

1.3.2 Water Scarcity:

Sultanate is categorized as arid country with low rain fall and high evapo-transpiration (ET). Rainfall varies from less than 50 mm in central Oman to more than 300 mm in north Oman Mountains. Ground water is the main source of water for both domestic and agriculture use. A large area of Batinah, the major agriculture region of the country, is facing a crucial problem of groundwater level decline and substantial deficit of fresh groundwater. Over pumping is the main reason for substantial deficit of fresh groundwater. Consequently, the fresh groundwater in the Batinah region has become saline due to sea water intrusion.

The consequences of aridity and high water use in Batinah have caused negative impacts on its agriculture environment viz. groundwater deficit and salinity. Impacts mainly include farm abandonment. These situations have led to a suggestion for imposition of desertification in Batinah (Al-Lawati, 1998 and FAO, 2008). On the other hand, limiting water resources in the country culminate in allocation of water to crops that yield higher and have better water-use efficiency.

At this point, it cannot be disputed that crop improvement is at the core of improved economic activity. Seemingly, this is highly depended on the availability of wide ranging germ plasm as well as efficient utilization of the same. Seemingly, alfalfa is distributed globally and grown in varied environments. The relative geographic expansion and adaptation enhances genetic variation and allows technicians to use diverse gene pools. Notably, alfalfa is an open pollinated, autotetraplod species. It is characterized by pronounced inbreeding depression and tetrasomic inheritance.

In light of above information, alfalfa, being the prime and preferred forage crop in Oman, could be explored for its improvement. This can be attained through plant breeding in a bid to develop high yielding genotypes that have high water-use efficiency and which are suitable under sprinkler irrigation. Notably, this would encourage sustainable cultivation. Proposed studies on the genetic aspects of WUE and related physiological traits would help in providing valid information for future alfalfa improvement program.

1.3.3 Diseases:

The following diseases have been reported in the farmer’s field.

• a) Crown rot and wilt: These severely attack the crop in the Southern plains (Salalah) where the crop succumbs to the diseases and becomes annual in nature. Waller and Bridge (1978) have recorded observations on the symptoms of the syndrome. The symptoms start with the wilting of the mature stems and girdling of the stems. The disease penetrates into the crown and results in the death of plant. Each cutting cycle experiences death of 10% of the plants and at the year end, the crop stand will be less than half of the original stand. The local sources indicated that the disease exists throughout the season. The disease would be severe during hot dry weather when frequent irrigations are provided. The causal organisms are suspected to be Melanospora zamiae L. and Fusarium salani L. as these were isolated from diseased crowns. However, disease could not be reproduced under laboratory conditions. It was concluded that disease could result from an interaction of pathogenic soil fungi with environmental factors.
• b) Leaf spot caused by Stemphylium globiferum is very common in the Interior on old alfalfa stands causing as much as 30% damage.
• c) Leaf rust caused by Uromyces striatus is common and locally severe in coastal areas, particularly on the young stands.
• d) Downy mildew caused by Peronospora trifoli results in some yield losses.
• e) Anthracnose on stems caused by Colletotrichum lindemuthianum is also observed on few stands.
• f) Witches’ broom, a mycoplasma disease has been reported in the farmers field recently (Al-Rawahi et al., 1998) and observed to be very serious in causing loss to the extent of 25% of green matter yield. It is characterized by formation of excessive short and spindly shoots with small, rounded and often pale green leaves. After 3 or 4 cuttings, the crown fails to grow and die, leaving empty patches in the field.
• g) Alfalfa mosaic virus was recorded in as many as 80% of the farms surveyed with 11-20% of disease incidence. The virus was found to be seed borne and hence disease incidence was higher in the fields where crop was planted with the seeds of infected plant stand (Anonymous, 1998).

Among the above diseases, the crown rot and wilt are very much endemic in Salalah plains and is of serious nature. Witches’ broom may emerge any time with surprise causing concern like the one noticed in Omani limes.  Other diseases however, occur sporadically depending upon conditions viz. temperature, relative humidity etc.

1.3.4. Insect Pests:

The following insect pests have been reported in the farmers’ field.

• a) Spotted alfalfa aphids (Theriphis maculata): occurs in damaging numbers.
• b) The potato leaf hopper (Empoasca fabae): causes stunting and yellowing.
• c) Lygus bugs (Lygus spp.):  cause serious damage to alfalfa seed production.
• d) Grass hoppers and alfalfa caterpillar: occasionally cause damage to the green matter.

1.3.5. Nematodes:

Omani alfalfa varieties like Qaryati, Batini and Interior were found to be completely resistant to Omani isolates of root knot nematode (Meloidogyne incognita) and the variety Dhofari was slightly susceptible, although there are indications that Qaryati does support very few low populations of Meloidogyne javanica (Sutherland, 1976).

1.4. Mechanization:

It is believed that local alfalfa cultivars are not amenable for mechanization, as they are required to be cut at ground level for regeneration of the crown.

1.5. Agronomic Characters:

In various agricultural systems, it is desirable to manipulate both height and tillering. Plant breeders have modified both apical dominance and plant height in the grass crops, but the genetic variability and behavioral details vary from crop to crop.

Ayers and Westcot (1976) conducted an investigation to identify the initial step in development of salinity-tolerant cultivars. They noticed that genetic variability for salinity tolerance could be identified within ecotypes of a species. They concluded that direct selection criteria could be based on growth or yield and provided important information for management decisions such as the salinity level where 50% growth reduction is expected.

Many researchers also reported that salt stress also results in a considerable decrease in the fresh and dry weights of leaves, stems, and roots (Hernandez et al., 1995; Ali Dinar et al., 1999; Chartzoulakis and Klapaki, 2000). It is now generally recognized that under the saline conditions the degree to which growth is reduced by salinity differs greatly with species and to a lesser extent with varieties within a species. The severity of salinity response is also mediated by environmental interactions, such as relative humidity, temperature, radiation and air pollution (Shannon et al., 1994).

1.5.1. Effects of Salinity on Physiologic Parameters

Multiple effects of salinity on plant physiology are related to enzyme activity, nutrient imbalance, membrane dysfunction, general metabolic processes, water relations and oxidative stress (Orcutt and Nilsen, 2000). Salinity stress drags out composite effects on plant mechanisms, which result in nutrient imbalance, water shortage and accumulation of toxic ions (Werner and Finkelstein, 1995). Many workers have investigated physiological responses of plants to salinity stress. However, effect of these elements of salinity stress on plants is still under question. Accumulation of toxic ions not only affects the plant metabolism under saline stresses but some other factors like pH and EC also play a role in retarding the plant growth which may be caused by high salt stress conditions.

Physiological adaptations to salt stress at the cellular level are the main responses amenable to molecular analysis and have led to the identification of a large number of genes induced by salt (Ingram and Bartels, 1996; Bray, 1997; Shinozaki et al., 1998).  Further there is physiological evidence to support the view that salt tolerance is a complex trait. Halophytes show a wide range of adaptations from the morphological to the biochemical adaptations that include the ability to remove salt through glandular activity (Flowers et al., 1986; Leach et al., 1990; Glenn et al., 1999; Tester and Davenport, 2003).Although control of ion uptake is exercised at the root, the ability to secrete ions has evolved into a successful strategy for salt tolerance. Some halophytes utilize salt-secreting glands to remove excess ions from their leaves (Thomson et al., 1988), reducing the need for a very tight balancing of ion accumulation and growth (Flowers and Yeo, 1986). Within less tolerant species, intra-specific variation in tolerance is also associated with variation in a wide variety of physiological traits (Yeo et al., 1990; Cuartero et al., 1992; Foolad, 1997; Wahid et al., 1997; Tozlu et al., 1999a, b).

1.6. Alfalfa’s Genetic Diversity

According to Radovic, Sokolovic and Markovic (2009), it is not only rich but also has a variable genetic base. Its performance is extremely well in different ecological conditions. The inherent adaptability can be used to explain why it tends to be assigned with a name in every ecological region that it is grown. For this reason, it can effectively adapt to different environmental conditions. The populations continually inbreed Alfalfa in order to develop a hybrid species. This eases adaptability of the species to different ecosystems without compromising productivity.

1.6.1 Genetic Mapping

Many agriculturally important variations such as productivity, quality and tolerance to biotic or abiotic stresses are controlled by polygenes. These complex traits are referred to as quantitative trait loci (QTLs), and it is challenging to identify QTLs based on only traditional phenotypic evaluation. Identification of QTLs of agronomic importance and its utilization in a crop improvement further requires mapping of these QTLs in a genome of crop species using molecular markers (Collard et al., 2005). The aim of many genetic mapping studies is to identify quantitative trait loci (QTL) that are responsible for phenotypic variation. Association mapping is a method for detection of gene effects based on linkage disequilibrium (LD) that complements QTL analysis in the development of tools for molecular plant breeding (Breseghello and  Sorrells, 2005). This approach identifies genotype-phenotype associations by identifying polymorphisms that are linked to functional alleles Myle et al., (2009). Tolerance to salinity is a quantitative or multigenic trait. DNA markers could be important in plant breeding if they are used to help in the selection of quantitative traits.

1.7.  Experimental Aims

The primary goal of this proposal is to investigate genetic diversity of indigenous and extic germplasm of alfalfa in respect of morphological, agronomic, physiological, biochemical and molecular characters

Phase I.  Evaluation of Alfalfa Accessions

• Planting the germplasm material to study morphological, agronomic, physiological, biochemical and molecular characters
• Classification of germplasm material based on morpho-physiological characters

• Molecular characterization of alfalfa accessions using microsatellite (SSR) primers.

2. 0 Materials and Methods

A pot study was undertaken to characterize and classify the genetic diversity among alfafa (Medico Sativa), explore the extent and pattern of both photypic and genetic variability in the collection, identify traits contributing to the overall variability, and classify the plants into groups based on some agro-morphological traits.

The seeds of 56 selected accessions of Alfalfa were sown on 7  June of  2012 in 10 pots for each accession . Each pot containing 5 plants for each accession. Spaced 15 centimeters apart within the pot. Constant monitoring and field evaluation is being done according to Li et al., (2009).

• 2. 1 Recording of Observations:

Data on agronomic characters and ion concentrations were recorded for all the fifty six selected accessions of Alfalfa.

• 2.2 Agronomic Characters:

The observations on agronomic characters such as plant height (cm) (which was measured from the soil surface to the terminal bud at 50 % bloom), number of leaves/ plant, leaf length (cm) and leaf width (cm) were measured on one randomly selected plants from each pot and expressed as mean. Data on days to 50% flowering were recorded on pot basis. Number of tillers and green matter (fodder) weight were recorded for each accessions. Dry matter weight was recorded for the samples in the laboratory after drying green matter in the oven at 70 C for 18-24 hrs (AOAC, 1984).

The first cut was taken after approximately 75 days after planting or when accessions showed flowering. Green matter yield from each accession was harvested and total fresh weight, dry weight, leaves dry weight, shoot dry weight, height, number of leaves and dry leaf/stem ratio were measured.

Characterization of these accessions will be done following the guidelines of IPGRI descriptors (1991). All data will be recorded in tables to ensure organization and uniformity.

• 2.3 Determination of Ionic Concentration in Plant Tissues:

The ionic concentrations namely P, K, Na, Cl, Ca and Mg were determined for the selected genotypes. The methods adapted for determination of the above ionic concentrations were determined following guidelines for plant analysis data that available in Reuter and Robinson (1986, 1997) and Jones et al. (1991).

• 2.4 Determination of Total Phosphorus   

In a glass beaker 25 g of ammonium molybdate are dissolved in 200m1 of distilled water. Sulfuric acid of 280ml conc. were then poured gently in to 400ml distilled water, the two steps were than mixed gently and dilute to 1 000ml.stannous chloride of 0.2g were than dissolved into 100ml v.flask, add 2 ml HCL conc. and dilute to 100m.

100 ppm standard are prepared by dissolving O.4393g of potassium phosphate KH2PO4 into 1 liter of distilled water to get 100 ppm. From 100 ppm 2 ml was taken into 100 ml flask to get 2 ppm.( 0.5ml to 5 ml) from a 100ml extraction was taken and pippate to 50 ml v. flask. Little of distilled water, 2 ml ammonium molybdate was added followed by addition of 2ml of stannous chloride to complete to the mark with distilled water ,the plant sample was tested using spectrophotometer  in a wave length of 700 nm.

• 2.5 Determination of Total Potassium, Na, Ca and M

Flame photometer machine are kept to warm for a couple of minutes.The 100ppm k std were than determined before reading of the samples.

• 2.6 Determination of Chloride (Cl)

Chloride is determined by silver nitrate titration, this is done by taking 10 ml of the extract into porcelain coercible, adding one drop of potassium chromate and swirl and then titrate with silver nitrate until the color is changed to light brown.

• 2.7 Physiological Traits

Concentrations of K+, Na+ and Ca2+ will be measured.

• 2.8 Phenotypic Data Measurements

Phenotypic characterization is critical to the classification of crop germplasm. The combination of both genetic and phenotypic characterization is essential in defining the plants’ variability of significant agronomic traits. During the investigation, ten phenotypic traits will be collected on all individual plants of each population.

 These will include;

1- Wilting Score (WS)

Scored from 0 up to 9. 0 with no symptoms of stress effect and 9 with all plants apparently dried. The wilting score will be crucial in determining the genetic and phenotypic mechanism of wilting in Alfalfa plants hence the wilting scores of the data pooled from different pots will be analyzed.

2- Average No. of Stems/Plant

The number of stems/plants of individual plants will be recorded directly following cuts.

3- Average Shoot Fresh Weight (g)

A random sample of fresh shoots will be chosen from each pot, placed into paper bags and weighed before being dried to determine the Average shoot dry weight.

4- Average Shoot Dry Weight (g)

The shoot dry weight will be established by measuring dried samples of the harvested plants which after being weighed as fresh samples.

The study will determine whether the phenotypic variables show any substantial differentiation among the population. The 10 phenotypic variables will also be compared with SSR markers to establish any correlation and to determine any significant variation between genetic and phenotypic differentiation. Additionally, patterns of differentiation for both phenotypic traits and SSR markers will be established and comparisons made.

3.0 Molecular Studies

3.1 SSR Analysis

Alfalfa is a perennial autotetraploid, allogamous species with 2n=4x=32 hence Simple Sequence Repeat (SSR) markers will be used in studying its genetic diversity in this study. SSR markers are considered efficient because they are highly polymorphic, are abundant in genome and tend to be co-dominant. Multiple studies have used SSR markers to estimate the genetic diversity in alfalfa with Thrall and Young (2000) presenting a range of tools for analyzing co-dominant markers in autotetraploids and Crochemore et al 1998 utilizing phenotypic traits as a valuable tool for studying and understanding genetic diversity among varied alfalfa populations.

Bulk DNA of several alfalfa plants will be obtained for each accession as recommended by Negri et al. (2000). Three grams of fresh young alfalfa leaves from four-week-old growing shoots will be collected from 10 plants in each accession, and immediately frozen in liquid nitrogen. Total genomic DNA will be extracted following (El-Kharbotly et al. 1998) with minor modifications (Al-Hinai, 2004). The PCR reactions will be performed as described by Falahati et al. (2007). Number of bands in each photograph will be determined and reproducible polymorphic bands from SSR analysis will be scored for presence (1) or absence (0) in each accession. A suitable software program will be used to estimate genetic diversities and genetic distances as well as achievement of a dendogram for the accessions. The values of genetic differentiation coefficients will be determined within the sampled Alfalfa population.

3. 2 Statistical Analysis

Statistical analysis of phenotypic data will be analyzed for each environment separately by one way ANOVA using appropriate software. The Pearson correlation coefficients (r) between the traits under both saline conditions will be calculated. Principal component analysis will also be performed on all traits with the traits being measured on different scales. Besides, the mean observation for each trait will be standardized prior to analysis to eliminate scale differences. Descriptive statistics such as the maximum, minimum, mean, deviations and phenotypic coefficient and variation for all the identified traits will be computed to estimate diversity. The ANOVA will be used to detect any differences among alfalfa populations for all traits which will thereafter be used to identify the traits which are the main source of variability and to explain any existing genetic diversity in germ plasm collections.

3.3 Phenotypic Data Analysis

The phenotypic variables will be analyzed using SAS following a mixed model analysis for variance. Prior to ANOVA, outliers will be removed using suitable studentized residual values and to conform to normality, a suitable statistical transformation will be carried out. Conventionally, ranked or non-normally distributed data should be analyzed using non-parametric tests based on ranks rather than continuous data.

3.4 Genomic DNA Extraction

Alfalfa accessions used for morphological character evaluation were used for the extraction of total genomic DNA. Thirteen varieties from America  were also included for DNA extraction.  Omani alfalfa accessions and American varieties were germinated and grown separately in pot. Young leaves of 3 weeks old seedlings of the alfalfa accessions were collected and stored immediately at -80 °C until further used for nucleic acid extraction. Total genomic DNA was extracted according to the CTAB procedure described by Doyle and Doyle (1987) with slight modifications. Young leaves were placed in 1.5 ml eppenddorf tubes containing a small amount of sterilized sand to facilitate the grinding process, 500 µl extraction buffer (1 % CTAB, 1.4 M NaCl, 0.1 M Tris- HCl (PH 8), 0.02 M EDTA (PH 8), 0.5 % (w/v) PVP, 0.1 % (v/v) ß-mercaptoethanol) was added and samples were ground using a mini pestle and mortar. Then 40µl of SDS (10%) was added to each sample. The samples were placed in the oven at 65 °C for 10 min and immediately placed on ice for 10 min. An equal amount of phenol: chloroform: Isoamyl Alcohol (25:24:1) was added to each tube, mixed gently. The tubes were centrifuged at 10,000 rpm at room temperature and the supernatant was then collected into new eppendorf tubes. An aliquot of 0.6 volume of isopropanol and 0.3M NaAc (pH 5.2) was added to the samples and placed at -20 °C over night. All samples were centrifuged at 10,000 rpm for 20 min at 4 °C and the supernatant were discarded. The DNA pellets were washed twice with 70 % ethanol followed by air-drying at room temperature. The DNA pellet was dissolved in 50 µl sterilized distilled water and stored at -20^oC until used. DNA samples for accessions within each region were prepared by bulking 5 µl of DNA extract. The concentration and purity of DNA was measured using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA).

3.5 Pre-Selective PCR

3.6 Post-Selective PCR

3.7 Fragment Analysis

3.8 Genetic Distance Estimates and Cluster Analysis

3.9 Structure Analysis

Structure analysis and relatedness relationships and marker-trait association analysis will be built from the data of greenhouse. Multivariate analysis will be used to determine the relationships between the multiple variables obtained in the study. The analysis will provide a better understanding of the structure of the collection, define the relationships among accessions, identify possible related groups and allow for the identification of the more relevant variables.

4.0 Results and Discussion

4.1 Conclusions and Further Work

The investigation is expected to reveal a wide phenotypic variability for the many of the assessed traits in the Alfalfa collection. Knowledge of genetic diversity in alfalfa populations originating from Omani (56) and the United States (13) will reveal any genetic diversity present in these populations. The PCA (Principal Component Analysis) will be adopted to separate the majority of populations into different clusters of germplasm. Positive traits of breeding will be assessed and identified in all clusters to help in future activities in alfalfa production, breeding programs and related scientific results globally. The evaluation, characterization and screening of the genetic resources and make up are considered critical in Alfalfa characterization and breeding, (Tucak et al. 2009). This is because the plant is distributed globally and grown in highly contrasting environments, a fact that has led to an extensive genetic variations and the existence of highly diverse genetic pools (Tucak et al. 2009).

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