Final Document

List of Contents

1.0 Introduction………………………………………………………………………..3

1.1 General Introduction………………………………………………………………3

1.2 Alfalfa Improvement in Oman- Prospects and Procedures………………………..3

1.2.1 Why Medicago Sativa? …………………………………………………………………………….3

1.2.2 Present Position……………………………………………………………………4

1.2.3 Alfalfa Growing Regions………………………………………………………..5

1.2.4 Alfalfa: A perennial Legume…………………………………………………….6

1.2.5 Improvement Objectives for Cultivar Development…………………………….8

1.2.6 Constraints in Alfalfa Improvement……………………………………………11

1.3 Problems related to Abiotic and Biotic Management Factors……………………12

1.3.1 Soil and Water Salinity…………………………………………………………12

1.3.2 Water Scarcity…………………………………………………………………13

1.3.3 Diseases…………………………………………………………………………14

1.3.4 Insect Pests……………………………………………………………………..15

1.3.5 Nematodes………………………………………………………………………15

1.4 Mechanization……………………………………………………………………15

1.5 Agronomic Factors……………………………………………………………….15

1.6 Effects of Salinity on Physiologic Parameters……………………………………16

1.7 Alfalfa’s Genetic Diversity………………………………………………………17

1.7.1 Genetic Mapping………………………………………………………………17

1.7.2 Experimental Aims……………………………………………………………18

1.7.3 Phase 1. Evaluation of Alfalfa Accessions…………………………………….19

2.0 Materials and Methods………………………………………………………….19

2.1 Recording and Observations…………………………………………………….19

2.2 Agronomic Characters……………………………………………………………20

2.3 Determination of Ionic concentration in Plant tissues……………………………20

2.4 Determination of Phosphorus……………………………………………………20

2.5 Determination of chloride……………………………………………………….21

2.6 Physiological Traits………………………………………………………………22

2.7 Phenotypic Data Measurements…………………………………………………21

3.0 Molecular Studies………………………………………………………………22

3.1 SSR Analysis……………………………………………………………………22

3.2 Statistical Analysis……………………………………………………………….23

3.3 Phenotypic Data Analysis………………………………………………………..23

3.4 Genomic DNA Extraction…………………………………………………………23

3.5 Pre-selective PCR…………………………………………………………………24

3.6 Post-selective PCR………………………………………………………………24

3.7 Fragment Analysis………………………………………………………………24

3.8 Genetic distance estimates and cluster analysis………………………………….24

3.9 Structure analysis…………………………………………………………………24

4.0 Results and Discussion……………………………………………………………25

5.0 Conclusions and Further work……………………………………………………25

6.0 References………………………………………………………………………..26

1. Introduction 1.1 General IntroductionIn Arid and semi arid lands, farmers depend solely on animal husbandry to cater for their livelihoods. In this regard, they practice large scale ranching in which they keep wide ranging livestock species. Limited rainfall greatly undermines their ability to explore other forms of farming effectively. For sustainable animal farming, they focus on plants that are highly productive. Certainly, forages of high digestibility and nutritive value enable them to reap optimally from these farming practices. This is at the core of their sustainable economic goals and objectives. Just like other farmers inhabiting arid and semi arid lands, farmers in Sultanate of Oman explore animal husbandry to sustain their livelihoods. This requires them to produce high quality forage for their animals in order to benefit optimally from the same. Alfalfa, scientifically, known as medicago sativa offers the best option for this. Michaud et al. (1988) stated that it is difficult to define precisely when and how alfalfa spread and reached various countries and areas. They explained that maritime trade was well developed in the eastern Mediterranean as early as 4000 B.C., which could have contributed to the spread of alfalfa and may have resulted in significant mixture of hybridization of ecotypes from widely separated regions.

Evidence of the ancient introductions of alfalfa into the Arabian Gulf is found in strongly marked characteristics of Arabian varieties which resulted from centuries of acclimatization in the arid region leading to the evolution of many unique local ecotypes of this crop. Relatively little use has been made of Middle Eastern alfalfa germplasm in formal breeding programs largely because variations among accessions from this region have not been systematically described or made widely available (Smith et al., 1995)

1.2 Alfalfa improvement in Oman – Prospect and Procedures1.2.1 Why Medicago sativa?

Alfalfa (Medicago sativa), the Queen of forage crops, forms an integral part of farm life in the Sultanate. Every farmer desires to grow it at least in small pieces of land depending on his holding to feed his goats, cattle or camels. Alfalfa plays a vital role in the agricultural economy of the country. In this regard, it accounts for almost half of the agricultural output (by value). It is the best quality feed for livestock as well as horses, contributing significantly to the quality of animal products. Nationally, it ranks top amongst the agricultural crops and has an annual production of an estimated 8.1 US dollars. Alfalfa forage is produced (harvested) throughout the year but it is higher during winter and low in summer.

It is a remarkable crop in comparison with others. Alfalfa is recognized as the most widely adapted agronomic crop, effective source of biological nitrogen (N2) fixation, energy efficient- crop to grow, important source of protein yield/ha and attractive source of nectar for honeybees. In addition to being an excellent source of vitamins and minerals, it is important for improving soil tilth (Barnes et .al., 1984). It is believed that alfalfa originated in South Western Asia (near Iran) but related forms and species are found scattered over central Asia as far north as Siberia. It was carried from Iran to Arabian Gulf, the Mediterranean countries and finally into Europe, America and Australia by traders, invading armies, explorers and missionaries as a valuable source of feed for horses and other animals. Evidence of the ancient introductions of alfalfa into the Arabian gulf is found in strongly marked characteristics of Arabian varieties resulted from centuries of acclimatization in the arid region. Few authors consider Arabian Peninsula as secondary center of diversity.

1.2.2 Present PositionThe Sultanate of Oman, being the third largest country in the Arabian Peninsula, has 73670.59 ha of agricultural land under cultivation of which fruits occupy a significant 50.33 % followed by perennial fodders (22.03 %), vegetables (8.41 %) and field crops (19.23 %). The total production, however, is shared highest by perennial fodders (53.09 %) followed by fruits (27.16 %), vegetables (15.67 %) and grain crops (4.08 %) (MoA, 2010). The fodder demand in the Sultanate is mostly met by the local production of alfalfa and Rhodes grass.

Table 1. Area and distribution of alfalfa in Oman1 (1995 data)

Sl.No. Region Cultivated Area (ha) Area under alfalfa (ha) % of alfalfa area

1.

2.

3.

4.

5.

6.

7.

8.

9. South Batinah

North Batinah

Sharqiya

Al-Wasta

Dhofar

Muscat

Dhahira

Interior

Musandam 11678

20643

7537

13

2822

3377

9421

6897

796 2411

3190

787

3

66

623

1662

1009

15 20.65

15.45

10.44

23.08

2.34

18.45

17.64

14.63

1.88

Total — 63184 9766 15.46

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

Alfalfa (Medicago sativa L.) forms an integral part of farm life in the Sultanate, as every farmer desires to grow it at least in small piece of land depending on his holding to feed his goat, sheep, cattle or camel, thus contributing about 11344 ha i.e. 15.40 percent of cultivated area (MAF, 1997). This feature seems to be common case throughout the Arabian Peninsula. It is grown widely in Batinah, Salalah plains, Interior and desert plains of Nejd. The region wise distribution cultivated area of alfalfa in the country is presented in Table 1.

1.2.3 Alfalfa Growing RegionsThe 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 legumeFrom 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 habit 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 through out 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, altho