The European Apis mellifera, Apis mellifera L. plays an of import function in pollenating blossoming workss and in conserving and advancing biodiversity and agricultural productiveness ( Klein et al. 2007 ) . One tierce of the entire human diet is dependent on insect-pollinated workss, predominately by the European Apis mellifera ( McGregor 1976 ) . A figure of harvests either depend chiefly on or are benefited by honeybee pollenation. In North America, for illustration, Apis melliferas pollinate no fewer than 90 harvests ( Partap 2011 ) ( In California, Prunus dulcis production is a USA $ 2 billion dollar industry, and is about wholly dependent upon honeybee pollenation ( Council 2007 ) . The pecuniary value of honeybee pollenation is estimated at about USA $ 15-20 billion dollars yearly ( Johnson 2010 ) .
Beekeeping is one of the longstanding traditional and environmentally friendly agricultural activities in Saudi Arabia. It is lending significantly for economic and societal development of the state through supplying alternate or extra income and supports of communities through the sale of honey and heightening agricultural productiveness, conserving environment and biodiversity. However bundle bee importings, of more than 100,000 settlements each twelvemonth, may advance successful pollenation and preservation of works vegetations in the land, it may increase an unwanted hybridisation of the local bee race with other bee races.
To increase honey production, apiarists in Saudi Arabia import more than 100,000 Colony every twelvemonth. However, more than 70 % of these settlements die out every twelvemonth due to abiotic emphasiss, chiefly due to high air temperatures ( Al Ghamdi 2005 ; Alqarni 2006 ) . The native Apis melliferas could go endangered due to one-year debut of Apis melliferas from different beginnings, familial dilution, eroding and it is predicted that within a few old ages, taking to the absence of pure familial resources will from the native bee. In this respects, the grade of familial dilution additions and presently, it is hard to find where pure native Apis melliferas could be found in the Kingdom of Saudi Arabia. In add-on, a few surveies were done sing to biological, morphometric, familial variableness, and individuality of native bee races in Saudi Arabia ( Alqarni 1995 ; Alqarni 2006 ; Alqarni 2011 ; under probe 2011 ) .
The native Apis mellifera race is the most cherished familial resource. The estimated figure of honeybee settlements in Saudi Arabia is between 700,000 to 800,000 owned by about 4,000 apiarists. Approximately, 200,000 of the above figure are the native races A. m. jemenitica ( Alqarni 2011 ) .
The lone native races of the Apis mellifera, A. m. jemenitica happening in Saudi Arabia, is unambiguously adapted to last to the hot air temperature conditions ( Nikolenko and Poskryakov 2002 ) . Therefore, it is really of import to conserve and better the native Apis mellifera through choice, genteelness, and preservation plan of native Apis mellifera populations. This will hold a great impact on bettering the apiculture industry of Saudi Arabia. It is hard to conserve and better the native Apis mellifera races without designation and appraisal of its grade of familial unity through analysis of morphometeric and familial characters. This designation and appraisal is an indispensable measure in conserving and bettering plans of the native Apis mellifera, A. m. jemenitica. Research is portion of a undertaking Funded by The National Plan for Science and Technology.
Assess the familial pureness of native Apis mellifera, A. m. jemenitica utilizing mitochondrial DNA molecular markers and atomic microsatellites techniques.
Designation of morpometric markers of Apis mellifera races including the native races correlated to the familial analysis in Saudi Arabia
Measuring the extent of cistron introgression of introduced Apis melliferas with A. m. jemenitica
Placing and positioning pure A. m. jemenitica distributions within Saudi Arabia.
Many morphometrical characters are used for a long clip to distinguish between Apis mellifera races and populations Morphometrics is the measuring and analysis of morphological characters and widely applied to analyze insect life history, physiology and systematic. Besides Morphometric steps both genotypic and phenotypic features. These include three classs: pigmentation, organic structure size, and venation of forewing angle, which represent 82.1 % of the entire fluctuation found in A. mellifera. The organic structure size factor is negatively correlated to temperature and positively correlated to altitude and represents about 45.5 % of the fluctuation. Pigmentation of the tergite represent about 18.6 % of the fluctuation, which is associated with temperature, height and rainfall ( Amssalu et al. , 2004 ) . Morphometric analysis has been used as a utile tool to measure races bounds in A. mellifera ( Ruttner and Louveaux 1978 ; Ruttner 1988 ) , and to analyze of familial variableness of Apis melliferas ( Garnery et al. , 1992 ; KekecoAYlu et al. , 2007 ; Miguel et al. , 2010 ) .
Historically, the categorizations of the Apis melliferas of the universe, 36 characters were identified. By using multivariate and stepwise discriminate analysis techniques, these characters were reduced to ten by ( Ruttner and Louveaux 1978 ) , 13 by, Amssalu et al. , ( 2004 ) or 11 characters by Andere et Al. ( 2008 ) for separation of Apis melliferas of different geographical beginning in Africa. Based on morphometric characters, the Apis mellifera, A. mellifera were classified into approximately 25 recognized races throughout the universe Ruttner 1988. These races were grouped into five major evolutionary line of descents as follows: the A ( Africa ) , M ( Western Europe ) , C ( South-Eastern Europe ) , and O ( Middle East ) , and Y ( Yemenitica ) line of descent ( Ruttner and Louveaux 1978 ; Ruttner 1988 ; Sheppard et al. , 1997 ; Franck et al. , 2001 ) . This categorization was mostly supported by mitochondrial DNA surveies ( Garnery et al. , 1992 ; Arias and Sheppard 1996 ; Franck et al. , 2000 ) , and microsatellite venue informations ( Miguel et al. , 2010 ) .
Word picture of native Apis mellifera
Native Apis mellifera race in Saudi Arabia is belongs to linage Y ( Franck et al. , 2001 ) . The first record of Apis mellifera in Saudi Arabia was described by Ruttner ( 1976 ) as a new races ; A. m. jemenitica, specimens collected from merely six locations within the Kingdom: two samples from Jazan, two samples from Riyadh and two samples from Alhasa ( Ruttner 1988 ) . The same races was besides found in Yemen ( Ruttner and Louveaux 1978 ) , Sudan ( Ruttner and Louveaux 1978 ; Rashad and El-Sarrag 1980 ; El Sarrag et al. , 1992 ) , Chad and Oman ( Engel 1999 ) , Somalia ( Ruttner 1988 ) , and Ethiopia ( Hepburn and Radloff 1998 ; Amssalu et al. , 2004 ) . However, the smallest Yemeni bee was recorded in Saudi Arabia ( Ruttner 1988 ) . It covers the western, southern and southeasterly parts of the Arabian Peninsula where the A. m. jemenitica adapted to environment ( Karpowicz 1989 ) .
Apis m. jemenitica is a little bee that happening in hot waterless zones of eastern Africa and the Arabian Peninsula ( Aqlan 1999 ; Engel 1999 ) . The morphometrics of A. m. jemenitica are placed in the lowest sector of the entire variableness, overlapping those of A. cerana ( Ruttner 1976 ) .The morphometric fluctuation in Yemeni bees may be due to inborn facets, environment, latitude and height. Besides recorded in Sudan races corresponds really closely to A. m. jemenitica every bit described as little organic structure size, slender and really xanthous ( Rashad and El-Sarrag 1980 ) . Harmonizing to morphometric analysis two differentiated populations of A. m. jemenitica were described from Yemen, but it belongs to the same races However, the population of Aden was smaller in size than other populations of A. m. jemenitica and deviated by 6.24-19.06 % from those recorded in many mentions of the same races ( Khanbash 1990 ) .
Because of restriction of morphometric analysis in measuring of familial diverseness, a figure of markers as fingerprinting methods have been development for the finding of genotype which are non susceptible to environment effects such as: allozymic fluctuation ( Contel et al. , 1977 ) . random amplified polymorphous DNA ( RAPD ) ( Williams et al. , 1990 ) , amplified fragment length polymorphism ( AFLP ) . Besides, whole genome microarray and individual nucleotide polymorphism ( SNP ) ( Whitfield et al. , 2006 ) . . These markers have been contributed significantly to analysis and understanding familial diverseness ( Behura 2006 ) and besides go a popular agencies for designation and word picture of works and animate being species ( Shaw et al. , 2002 ) .
Mitochondrial DNA ( mtDNA ) Marker
The ( mtDNA ) is a closed-circular, double-stranded molecule, runing in size from 15 to 20 kilobits ( Wolstenholme 1992 ) . Analysis of mtDNA fluctuations have been used to distinguish between five line of descents of Apis melliferas ( Garnery et al. , 1992 ; Franck et al. , 2000 ; Palmer et al. , 2000 ; Sheppard and Smith 2000 ) . Mitochondrial sequences are known to be utile in know aparting among honeybee line of descents and fluctuation between and within honeybee population ( Cornuet et al. , 1991 ; Smith et al. , 1991 ; Garnery et al. , 1992 ; Moritz 1995 ) . All Apis melliferas have a non-coding sequence, which appears to be alone to bee genus Apis sp, located between the mitochondrial COI and COII cistrons ( Cornuet et al. , 1991 ) . In the Apis mellifera, as in most animate beings, chondriosomes are inherited merely from the female parent, so all persons in a settlement portion the same mitochondrial DNA ( Crozier and Crozier 1993 ) . It reveals more than 50 haplotypes in line of descents A and M ( Garnery et al. , 1992 ) but merely six haplotypes in line of descent C ( Franck et al. , 2001 ; Kandemir et al. , 2006 ; Ozdil et al. , 2009 ) .
Restriction fragment length polymorphisms ( RFLPs ) utilizing mtDNA has been utile marker for the survey of geographic beginning and familial fluctuation in Apis melliferas ( Hall 1990 ; Hall 1991 ; Arias and Sheppard 1996 ; Alippi et al. , 2002 ) . The most widely used mtDNA marker gave fluctuation was in the intergenic part between the COI and the COII cistron in Apis mellifera ( Garnery et al. , 1992 ) . Mitochondrial COI-COII intergenic part of the Apis mellifera have been utile in distinguishing evolutionary line of descents and groups of races ( Cornuet et al. , 1991 ; Hall 1991 ; Garnery et al. , 1992 ; Franck et al. , 2000 ) . So the analysis of mitochondrial COI-COII will be indispensable to distinguish between the native Apis mellifera and other Apis melliferas races.
Simple sequence repeated ( SSR ) or microsatellites markers
SSR markers are tandem repetitions interspersed throughout the genome and they can be amplified utilizing primers. SSR has been successfully used to analyze familial fluctuation within populations of the same species, such as Apis melliferas ( Brown et al. , 1996 ) . This marker motherly and paternally inherited, which enables hybridisation and cistron introgression surveies, leting the differentiation between homozygous and heterozygous persons, and normally used to separate Apis mellifera line of descents on the footing of morphometric ( Ruttner 1988 ) and mtDNA ( Smith et al. , 1991 ) , and races ( Hall 1986 ; 1990 ; Hall and Smith 1991 ) . Microsatellite markers from individual and multiple venue particular to eastern European, western European, and African bees have been used to qualify Old World European and African Apis mellifera populations ( Estoup et al. , 1993 ; Estoup et al. , 1995 ) .
In the present probe, mtDNA markers and SSR markers combination will be used to analyze the familial diverseness of Apis melliferas from vicinities in Saudi Arabia. Additionally, it will be analyze morphometrically variable populations for familial construction. This facet of the survey will concentrate on non-migratory settlements along Sarawat Mountains and Tehama Plains sites that have been kept isolated from foreign Queenss debuts, so that it may good reflect the hereditary familial features of Apis melliferas vicinities in western and southwesterly Saudi Arabia. In addotion, this survey will measure the pureness of Saudi non-migratory settlements of A. m. jemenitica.
Materials and Methods
Initial studies will turn up native Apis mellifera populations in the Kingdom of Saudi Arabia, concentrating on current distribution of A. m. jemenitica. The survey will cover all parts of Saudi Arabia. Representative samples will be collected from all parts particularly from countries of active apiculture. Stratified sampling of three degrees ; part, state and locations will be used ( Bee et al. , 2010 ) .
Designation of parts, states and locations
The Saudi Arabia divided geographically into 13 parts harmonizing to the current functionary administrative division. Samples will be collected from each part harmonizing to stratified Sampling Technique. So the samples will be classified to three strata: high denseness, medium denseness, and low denseness populations harmonizing to the figure of native Apis mellifera settlements in Saudi Arabia or in the part ( Alqarni 2011 ) . The Quota of collected samples will be 42 % high denseness countries, 44 % center, and 14 % little of the entire samples. The Quota will be distributed every bit for all of the parts. The Quota of each of the parts will be 21 % for high denseness, 11 % medium and little 2 % of the entire samples ( Appendix 1 ) .
It will be interview with apiarists in every part of state to obtain vicinities of active bee houses within the states. Subsequently, it will concentrate on part where isolated non-migratory bee settlements ( A. m. jemenitica ) occur and countries with imported Apis melliferas that have existed on the same site for at least four old ages or moved within the part for more than 100 kilometers in any way. In extra samples will be collected from imported bees to measure possible cistron flow between A. m. jemenitica and imported Apis mellifera settlements. Sample size
The figure of honeybee settlements in Saudi Arabia are estimated between 700,000 to 800,000 owned by about 4,000 apiarists. Approximately, 200,000 of the above figure are the native races A. m. jemenitica ( Alqarni probe 2011 ) . The representative sample of each population will be about 200 sample, which represents about. 0. 1 % of the entire figure of native Apis mellifera settlements as shown in Appendix ( 1 ) .
Sample aggregation Method
About 100 grownup worker bees will be indiscriminately collected from each hive. After that, samples will be divided into two parts ; the first portion will be used for morphometric analysis and the 2nd portion for familial analysis. The first portion will be treated by hot H2O ( to obtain to the full stretched proboscis of the dead bees ) , put it into little plastic bottles, preserved in 70 % ethyl alcohol with glycerol and be labeled ( Ruttner and Louveaux 1978 ) . The 2nd portion will be instantly preserved in eppendorf tubing by submergence absolute ethyl alcohol, Change the ethyl alcohol after 12-l6 H, so stored at -20 A°C during the aggregation and so transported to the research lab to be stored at A-80A°C until used for DNA extraction ( Hall 1995 ) .
Placing and positioning
During the collection, a GPS unit will be used to find lift ; map co-ordinates for each hive of native Apis melliferas and converted to a bed of GIS. Program Map will be used and study to find locations of Apis mellifera urtications. Besides, all necessary environmental information will be recorded and if imported Apis mellifera settlements are nearby.
Ten workers from each settlement will be dissected under binocular microscope and mounted as follows: dehydrated in intoxicants, cleand in xylol, topographic point on slides and fixed utilizing Canada balsam ( Adsavakulchai et al. , 1999 ) . The morphometric characters will be processed utilizing the computing machine package ; ( UTHSCSA Image Tool package, University of Texas Health Science Center San Antonio Image Tool Ver. 3.00 ) under declaration up to 2400 dpi. ( Wilcox et al. , 2002 ) .
For each single bee, 20 morphometric characters that have high discriminatory power will be measured following ( Ruttner 1988 ) . These characters include: Length of proboscis ( 5 ) . Length of thighbone ( Fe ) ( 6 ) , Length of shinbone ( Ti ) ( 7 ) , Length of tarsus ( Ta ) ( 8 ) , Longitudinal diameter of tergite 3 ( T3 ) ( 13 ) , Longitudinal diameter of tergite 4 ( T4 ) ( 14 ) , Longitudinal diameter of sternite 3 ( 15 ) , Longitudinal of mirror sternite 3 ( 16 ) , mirror transversal sternite 3 ( 17 ) , Distance between mirror ( 18 ) , Longitudinal dimeter of sternite 6 ( 19 ) , Transversal of sternite 6 ( 20 ) , flying length ( 21 ) , flying breadth ( 22 ) , Number of hamuli ( 42 ) , Cubital index a/b ( 2728 ) , flying angle B4 ( 22 ) , flying angle N23 ( 30 ) , flying angle O26 ( 31 ) , Pigmentation of abdominal tergite 2 ( 32 ) , pigmentation of abdominal tergite 3 ( 33 ) , pigmentation of abdominal tergite 4 ( 34 ) .
Reference informations of Apis mellifera races will be obtained from the database of the Institut fur Bienenkunde, Oberursel in Frankfurt, Germany. Which include A. m. jemenitica, A. m. carnica, A. m. syriaca, A. m. ligustica and. A.m meda ( .
Data will be subjected to principal component analysis and discriminate analysis utilizing the SPSS statistical package. ( Kandemir et al. , 2006 ) .
Deoxyribonucleic acid extraction
Entire nDNA will be extracted from the thorax of a individual grownup worker per settlement utilizing a DNeasy Blood & A ; Tissue Kit ( Qiagen, Valencia, California ) . The same sample will be used for both mtDNA and microsatellite venue analyses. The protocol followed will be harmonizing to the maker ‘s instructions for carnal tissue ( Appendix 2 ) .
PCR elaborations ( mtDNA analysis )
mtDNA analysis will be performed on the intergenic part between the COI and COII cistrons. This part will be amplified utilizing Deoxyribonucleic acid from merely one bee worker per settlement by utilizing PCR and primers E2 5 ‘ -GGCAGAATAAGTGCATTG – 3 ‘ , H2 5’- CAATATCATTGATGACC-3 ‘ ( Cornuet et al. , 1991 ; Garnery et al. , 1992 ) . Each sample reaction will be performed in 25 I?L incorporating 2.7 I?L of Taq buffer ( Promega, Madison, WI ) , 1.5 millimeter MgCl2, 25 nmol of each dNTP, 25 pmol of E2 and H2 primers, 11.8 I?L of distilled H2O, 0.5 I?l ( 5 U/I?l ) of Taq polymerase ( Promega ) , and 1I?L of DNA infusion. The PCR conditions for all reactions will be one 2 min rhythm at 95 °C, 30 rhythms of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s and a 5 min rhythm at 72 °C in a GeneAmp 9700 thermocycler ( Applied Biosystems ) . To find the size of the amplified part, a 6 I?L aliquot of PCR merchandise electrophoreses on a 1.4 % agarose gel. And visualise with ethidium bromide in UV visible radiation with aA Gel DocA imager ( syngene ) .
Digestion with limitation enzymes
5I?L from PCR merchandises from each sample will be digested for 3 H at 37 CA° with 0.2 I?l of BSA ( Promega ) , 2 I?l of enzyme buffer ( Promega ) and 1 I?l of limitation enzyme ( Promega ) ; the concluding volume will be adjusted to 20 I?l by adding unfertile distilled H2O ( Shahera Zaitoun et al. , 2008 ) . The restricted Deoxyribonucleic acid fragments will be separated on a 10 % polyacrylamide gel or 2 % Metaphor agarose and stained with ethidium bromide. The limitation fragment will be scored as present ( PCR merchandise cut ) or absent ( PCR merchandise non cut ) , based on the visual image of a two-band form or one-band form on the gel harmonizing to ( Garnery et al. , 1998 ) .
Deoxyribonucleic acid sequencing
Colonies will be classified harmonizing to their mtDNA haplotype. Each new haplotype sequenced to corroborate its singularity and individuality with haplotypes published by ( Franck et al. 1998 ) . The PCR merchandises will be purified utilizing QIAquick Gel Extraction Kit ( QIAGEN ) . Sequences of haplotypes will be aligned with published sequences of 44 different haplotypes of honey bees manually with Cluster package ( Thompson 1999 ) .
Polymerase concatenation reaction ( PCR ) will be amplified eight specific microsatellite venue by Eight braces specific primers viz. ( A7, A24, A28, A88, A113, B124 ( Estoup et al. , 1995 ) , Ap43 ( Garnery et al. , 1998 ) , Ap81 ( Solignac et al. , 2003 ) . These venues have been shown to be good placeholders for measuring familial fluctuation in Apis melliferas at the population degree.
PCR will be amplified in 20 AµL reactions incorporating 1 AµL extracted DNA, 1A- reaction buffer Promega ( Madison, WI ) , 3 millimeter dNTP mixture, 1.0-4.0 millimeter primer, , 1.5 units Taq polymerase ( Promega, Madison WI ) and the MgCl2 1.5 millimeter. The unvarying PCR will be used for all reactions: one rhythm 2 min at 95 °C, 30 rhythms of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s and a 5 min rhythm at 72 °C. PCR merchandises will be visualized on 2 % Metaphor agarose stained with ethidium bromide. Microsatellite fragment sizes will be scored by comparing the length of the PCR fragments to the standerd 100 bp ( Qiagen ) .
For Mitochondrial analysis indifferent estimations and standard divergences of cistron diverseness of mtDNA ( Nei and Tajima 1981 ) and appraisals of the population subdivision will be documented utilizing the Arlequin package bundle ( Schneider et al. , 1997 ) .
Microsatellite allelomorph sizes will be scored by comparing of the lengths of the PCR merchandises to the stander 100 lead marker ( qiagem ) . The figure of allelomorphs per venue ( n ) and expected heterozygozity ( He ) will be estimated with GENEPOP v3.4 package ( Nei 1973 ; Raymond and Rousset 1995 ) . Hardy-Weinberg trial will be performed by the Markov concatenation method for every venue in each vicinity ( Guo and Thompson 1992 ) . The deferent bee houses will be tested for population distinction with the Fisher exact trial ( Raymond and Rousset 1995 ) . The cyto-nuclear disequilibrium will be analyzed with the same trial ( Asmussen et al. , 1987 ) .
Research Time Line
Type of work
preparing and ordination of stuffs and equipments
Surveying and sample aggregation
Entire DNA extraction and pureness and concentration of DNA determined
PCR elaboration for RFLP
Type of work
PCR elaboration for Microsatellite
composing and publication