Odeigah and Osanyinpeju ( 1998 ) were non far from world when they stated that many autochthonal African harvest workss which have been used for 1000s of old ages have late been neglected, due to the debut of new alien harvests. These ‘lost harvests ‘ as they are sometimes referred to, hold the potency of bettering the nutrient security position of developing states but require scientific attending in order to re-introduce them into farming system. Bambara Indian potato ( Vigna subterranean L. Verde ) is an autochthonal tropical African harvest. Prior to the debut of the American Indian potato ( Arachis hypogaea L. ) into Africa, bambara Indian potato was booming as a major harvest. Consequently, it is presently classified as one of the universe ‘s underutilised harvest species. The likely Centre of beginning of bambara Indian potato is in West Africa with North-Eastern Nigeria and Northern Cameroon believed to be the exact topographic points of beginning. The harvest is besides cultivated in Asia, Australia and Latin America.

Bambara Indian potato is herbaceous, intermediate, one-year, self-pollinating works of the household Leguminosae, subfamily Papilionoideae. The works has bunched foliages originating from branched stems that signifier a Crown on the dirt surface. Its podding wont is similar to that of A. hypogaea in that the pale xanthous flower chaff bends downwards after fertilisation, forcing the immature cod into the dirt, where it develops and matures ( Amadou et al. 2001 ) . For optimum growing and output, the works requires a frost-free period of 3.5 months, bright sunlight ; mean day-to-day temperatures of 20-280C and 750-900mm of equally distributed rainfall ( Khonga et al. , 2003 ) .

Bambara Indian potato has many nutritionary, medicative and environmental advantages over other harvests in the same household. This grain leguminous plant is by and large cultivated by adult females subsistence husbandmans in tropical bomber Saharan Africa utilizing unimproved techniques. It is comparatively immune to biotic and abiotic emphasiss and is said to be the 3rd most of import leguminous plant after Indian potato and black-eyed pea ( Vigna unguiculata ) ( FAO, 2001 ) . Bambara Indian potato contributes soil N for other harvests by repairing atmospheric N through mutualism with Rhizobium bacteriums and is hence good in harvest rotary motions and intercropping. Chemical analyses of bambara seed have shown that it contains 32.72 % of entire indispensable amino acids and 66.10 % of entire non-essential amino acids. Lysine is the major indispensable amino acid and represents 10.3 % of the entire indispensable amino acid. Bambara Indian potato is besides a good beginning of leucine and contains a sensible sum of phenylalanine, histidine and valine ( Ntundu et al. 2004 ) .

Despite the importance of bambara Indian potato as a nutrient leguminous plant in traditional agriculture systems, limited genteelness attempts have been made to better this harvest. Small information is available about the extent of familial diverseness among bambara landraces, for long term preservation and betterment. The Indian potato is still cultivated utilizing landraces with low and unpredictable outputs. Drabo et Al. ( 1995 ) reported that universe bambara Indian potato production was about 330 000 T with 45-50 % of the entire universe production coming from West Africa. Under assorted agro-ecological zones, bambara Indian potato landraces have been reported to exhibit different grain output potencies depending on the genotype and to a great extent influenced by environmental conditions like temperature and rainfall. For illustration, Khonga et Al. ( 2003 ) have reported that mean outputs of bambara Indian potato landraces could run between 13.7 kilograms ha-1 to 177.3 kilograms ha-1 depending on the genotype.

Till day of the month, bambara Indian potato is cultivated from unimproved local landraces exhibiting low output associated with hapless sprouting per centum and hapless harvest constitution. Ouedraogo et Al. ( 2008 ) study that research on bambara Indian potato has been really limited compared with probe made on sorghum, millet, maize, peanut and black-eyed pea. Traditionally, morphological, ecological location and agronomic traits have been used for designation of different genotypes of the harvest ( Massawe et al. , 2005 ) . Attempts to better the output potency of bambara Indian potato is plagued with restrictions originating from unequal informations on the familial variableness and diverseness of the harvest. Recently, scientists have begun to roll up agronomic, physiological and molecular cognition about the harvest aimed at cut downing the spread between current on-farm output and its familial potency.

The usage of molecular marker to analyze familial diverseness of Bamabara Indian potato has been reported. Techniques such as RFLP ( Restriction Fragment Length Polymorphism ) , RAPD ( Random Amplified Polymorphic DNA ) , AFLP ( Amplified Fragment Length Polymorphism ) have been reported ( Massawe et al. , 2003 ; Amadou et al. , 2001 ) . In this survey microsatellite or simple sequence repetitions ( SSRs ) were used to analyze the familial diverseness of bambara Indian potato. SSRs are reported to be extremely polymorphous and co-dominant by Matthews et Al. ( 1999 ) . Knowledge about the diverseness of these landraces would supply breeders with specific information required in controlled genteelness of genotypes that are adapted to specify environmental conditions. It is besides hoped that the consequences of this survey will ease the certification of the familial diverseness available to bambara Indian potato breeders and therefore help the reintroduction of the harvest into farming systems.

2.0 AIMS AND HYPOTHESIS

This survey aimed at finding the familial diverseness of 24 landraces utilizing microsatellites or simple sequence repetitions ( SSRs ) . The survey was based on the hypothesis that bambara Indian potato landraces cultivated in different agro-ecological zones are genetically unrelated. There is therefore the demand to find the heterogeneousness or homogeneousness of these landraces.

3.0 MATERIALS AND METHOD

3.1 Plant stuff

Twenty-four landraces of V. subterranea from different African and Asiatic states were kindly provided by the Plant and Crop Science Department, University of Nottingham, Sutton Bonington Campus. These included 8 landraces from Indonesia, 3 landraces from Namibia, 2 each from Cameroon and Botswana, 1 each from Swaziland, Ghana, Zimbabwe, Zambia, Nigeria, Tanzania, Mali, South Africa and Malawi ( Table 1 ) . Leaf samples from each landrace were collected for DNA extraction and analysis.

Table 1. Bambara Indian potato landraces used and their beginning

No.

Name of Landrace

GEOGRAPHICALORIGIN

No.

Name OF LANDRACE

GEOGRAPHICAL ORIGIN

1

Uniswa Red

Swaziland

13

TVSu 999

Rhodesia

2

BCL

Dutch east indies

14

TVSu 747

Northern rhodesia

3

S19-3

South west africa

15

TVSu 569

Cameroon

4

BC

Dutch east indies

16

TVSu 610

Nigeria

5

Nav-4

Ghana

17

Dod Red

Tanzania

6

DipC

Botswana

18

SB16-5A

South west africa

7

BHC

Dutch east indies

19

Tiga Nicara

French sudan

8

Gigahertz

Dutch east indies

20

Ahm 968

South west africa

9

GH

Dutch east indies

21

VSSP 6

Cameroon

10

Bohrium

Dutch east indies

22

AS 17

South Africa

11

GCL

Dutch east indies

23

Gab C

Botswana

12

GHC

Dutch east indies

24

Mal 3

Nyasaland

3.2 DNA Extraction

Young leaf tissue ( 2-5 g ) was collected, rapidly stop dead in liquid N, transferred to lyophilise ( 350 µl of lysis solution [ Part A ] and 50µl of lysis solution [ Part B ] ) , and land with howitzer and stamp. Of this stuff, about 0.3-0.4 g was used to pull out DNA utilizing the Sigma GenEluteTM Plant Genomic DNA Miniprep Kit process. DNA concentration and visual image was determined by comparing with lambda DNA criterions on 1 % agarose gel stained with ethidium bromide ( 0.5 µg µL-1 ) as shown in figure 1.

Fig 1: Picture demoing DNA nowadays in extracted samples.

3.3 PCR Amplification and DNA Analysis

PCR was carried out in 20-?l volume in a mixture incorporating 14.4µl unfertile H2O, 2 µl MgCl2 PCR buffer, 0.5µl frontward primer, 0.5µl contrary prime, 0.4µl dNTPs, 0.2µl Taq polymerase and 2µl templet DNA. DNA elaboration was performed as follows: 180 s at 94 & A ; deg ; C followed by 35 rhythms with ( I ) 60 s at 94 & A ; deg ; C, ( two ) 60 s at 60 & A ; deg ; C, and ( three ) 120 s at 72 & A ; deg ; C. After the concluding rhythm, the samples were held for 600 s ( 10 proceedingss ) at 72 & A ; deg ; C ( modified Mayes ‘s Lab Protocol ) . Amplification merchandises were analyzed by cataphoresis in agarose ( 2 % agarose gel for PCR merchandises, 1 % for genomic DNA ) gel in a TBE buffer. Permanent records were obtained by snaping ethidium bromide-stained gels under UV visible radiation. The 2-Log DNA ladder was used as a standard molecular weight size marker. Figure 2 shows a image of the PCR merchandises.

Fig 2. Merchandises of our polymerase concatenation reaction

Capillary cataphoresis was performed utilizing Beckman CEQ 8000 capillary sequenator. Eight random microsatellites of 20-base brace sequences that yielded consistent and polymorphous merchandises were used ( Table 2 ) . These were selected based on microsatellites sizes and assorted such that polymorphous sets of specific sizes could be amplified by several primers without any convergence.

3.4 DATA ANALYSIS

Following satisfactory duplicability of the separate DNA extractions and elaboration procedure, the information was analysed utilizing multivariate statistical bundle MVSP version 3.13q. Based on a process described by Kloda et Al. ( 2008 ) a matrix was constructed numbering each single landrace sample as a individual instance and each microsatellite allele as a variable, scored as present ( 1 ) or absent ( 0 ) ( see appendix ) . The composite landraces were presented as a individual row. A matrix of allele frequences was besides constructed, stand foring the frequence of each allelomorph for each landrace. The informations sets were explored utilizing Principal Coordinates Analysis ( PCO ) implemented through the Multivariate Statistical Package ( MVSP ) . This ordination method harmonizing to the writers makes no premises about the distribution of the random variables. Furthermore, Euclidian distance was chosen in penchant to other distance steps, as it does non category common absence of an allelomorph as a shared feature, and was hence judged to be most appropriate in the present survey, which included extremely polymorphous microsatellite informations characterizing bambara Indian potato landraces. A dendogram was generated based on Nei and Li ‘s similarity coefficient utilizing Unweighted Pair Group Method with Arithmetic Mean ( UPGMA ) from a matrix of presence and absence of each allelomorph.

Table 2: List of the 20-base brace nucleotide primers used and their sizes

Primer name

Primer Length

Tm ( oC )

Amplified

Temp

Size Rnge ( bp )

Primer 1

20

57.3

Yes

60.2

254-256

Primer 2

20

59.4

Yes

59.7

173-175

Primer 4

20

55.9

Yes

59.7

200-202

Primer 5

20

57.3

Yes

59.7

207-208

Primer 16

20

55.3

Yes

60.2

185-189

Primer 19

20

53.2

Yes

60.2

134-269

Primer 27

20

55.3

Yes

60.2

226-228

Primer 30

20

55.1

Yes

60.2

246-249

4.0 Consequence

Consequences from the bunch analysis are summarised as a dendrogram ( Figure 3 ) , demoing the relationships among landraces as determined by the similarity between markers. Cluster analysis revealed that familial distances between all braces of the 24 bambara Indian potato landraces varied from 0.22 to 1.00, with a entire mean familial distance of 0.75. By and large, there were two chief bunchs and bunch 2 was farther divided into three sub-clusters picturing changing grades of familial diverseness between the landraces. Cluster one was made up of 2 landraces viz. TVSu 999 from South Africa and Uniswa Red from Swaziland. Cluster 2 was made up of the staying 22 landraces. There were by and large close relationships between landraces from the same location ; the sub-clusters of bunch 2 were observed to be grouped harmonizing to their geographical beginnings with landraces from Asia, West and Southern Africa each by and large grouped together. GC and BHC, both from Indonesia were seen to be the same landrace but cultivated in different parts of the state with different names. With exclusion of primer 30 which did non bring forth clear stria forms, all the staying seven primers confirmed this homogeneousness ( Refer to appendix ) . Similarly, BH, GH and GCL exhibited similarity coefficient of 1 bespeaking that these landraces are besides the same with different names harmonizing to six microsatellites baring primer 4 and primer 30 which was non scored for. A similarity coefficient of 0.92 between the two sub bunchs of landraces from Indonesia indicates that these are so really closely related. A really interesting observation was the realization that AS17 from the Republic of South Africa and BCL signifier Indonesia were the same landraces as amplified by six primers but for primers 16 and 30. These two landraces were observed to be closely related to the Southern African sub-cluster of landraces with a similarity coefficient of over 0.92. This observation is made clear from the PCO spread secret plan ( figure 4 ) where BCL is clearly seen to be portion of the Southern African group and distinctively off from the Asian group of landraces. Furthermore, Dod Red of Tanzania ( East Africa ) happens to be closely related to Asian landraces than to the African 1s whereas VSSP 6 of Cameroon and SB16 5A of Namibia besides exhibited close relationship ( refer to calculate 3 and 4 ) .

Fig 3: UPGMA dendogram of 24 bambara Indian potato landraces with Nei ‘ and Li ‘s similarity coefficient

Fig. 4: Euclidian PCO analysis for 24 bambara Indian potato landraces. Axes 1 and 2 represent 28.70 % and 17.40 % of the fluctuation nowadays, severally, based on an analysis for all the landraces.

5.0 Discussion

The aim of the present work was to analyze the sum of familial diverseness and to set up familial relationships among contrasting bambara Indian potato landraces from different turning parts in Africa and Asia utilizing microsatellite markers. Grouping of the bambara Indian potato landraces harmonizing to geographic beginning indicates considerable familial divergency likely due to different turning environments. Landraces used in this survey represented the best possible scope of morphological variableness and geographical beginning available in the cultivated signifier of bambara Indian potato.

In entire, 150 different allelomorphs were detected across the 7 microsatellites genotyped on 24 landraces from Africa and Asia. The PCO analysis in Figure 4 suggests three wide groups: they include landraces from West Africa, Southern Africa and Asia. Although some landraces overlapped, these were by and large limited with 46.10 % of the molecular fluctuation explained by the first two axes. Although, the survey demonstrates that there is considerable diverseness in landraces of bambara Indian potato, some landraces nevertheless, produced indistinguishable electropherograms proposing that these landraces are in fact the same genotype which due to their geographical beginning have been given different names. The considerable high degrees of familial polymorphism observed in this survey indicate that most of these landraces are extremely diverse from one other. Massawe et al. , ( 2002 ) have reported that the major contributory factor to high degrees of familial diverseness in many species is the nature of their genteelness systems with out-crossing species holding higher diverseness than self fertilized species. Although bambara Indian potato is mostly Self-fertilising, the writers reported that cross pollenation occurs and is aided by emmets.

Whereas some landraces were shown to be of the same genotypes, there was close relationship between some of the landraces in this survey, presumptively because they were collected from similar locations. From the SSR analyses these landraces may hold been derived from the same location. For illustration TVSu 999 from Zambabwe and Uniswa red from Swaziland are closely related and may be so the same. Landraces have local names based on testa coloring material and their cultivation site. Such an informal taxonomy may take to one landrace holding more than a individual name as a effect of seed debuts from other topographic points. This could be farther attributed to the fact that these two states are neighbours and there is informal motion of people across boundary lines and free trade ( Oucho and Crush, 2001 ) . Generally some of the landraces from South African states, West African states and Indonesia, revealed close familial affinities to members from their several topographic point of beginning. An interesting point is that AS 17 and BCL, originally from South Africa and Indonesia severally, were seen to be the same. Introduction of seeds from one location to the other by harvest breeders could be the possible account of this happening. Besides, Azam-Ali and Squire ( 2002 ) reported that bambara Indian potato that is grown in Indonesia is a consequence of the slaves who took the harvest to Surinam and was farther dispersed to countries such as South and Central America, India, Indonesia, Malaysia, the Philippines, Sri Lanka and parts of northern Australia. Additionally, the universe is now said to be a planetary small town with free motion of people further heightening the possibility of scattering of harvests across continents.

The grouping of SB-16 5A from Namibia with VSSP 6 from Cameroon and its subsequent grouping with the West African landraces likely indicates that SB-16 5A besides originated from Cameroon or one of the West African provinces. This is likely because Cameroon forms a passage between West and South Africa and it is possible that familial stuff was moved from one state to the other. The observation that, based on the UPGMA analyses, the lone East African landrace examined ( Dod Red from Tanzania ) was non grouped with the African types harmonizing to its putative geographical beginning but was instead closely related to the Indonesian landraces could besides be attributed to the motion of seeds by husbandmans and works breeders. Amadou et Al. ( 2001 ) reported similar findings where it was suggested that familial stuff might hold been moved from one state to another.

Four landraces from West Africa, six from Asia and seven from South Africa were clustered together demoing that SSRs are capable of uncovering really near relationships. The familial variableness among groups of landraces suggests that landraces may consist a mixture of genotypes ( Massawe et al. , 2002 ) and this will hold deductions on landrace stableness across variable environmental conditions. In applied genteelness, familial distances have been said to supply forecasters for hybrid vigor ( Ntundu et al. , 2004 ) . Our consequences for bambara Indian potato landraces indicated short familial distances between some landraces and comparatively longer distances between others. This cognition would supply breeders with specific information required in controlled genteelness of genotypes in order to maximize intercrossed energies. For illustration, to maximise familial diverseness, unreal hybridisation should be done between landraces that are genetically distinguishable ( with low familial similarity values ) , such as Uniswa Red and VSSP 6 with Nie and Li ‘s similarity coefficient of merely 0.22 as revealed by our PCO analyses.

6.0 Decision

Knowledge about the diverseness of harvests can be exploited by breeders to command the genteelness of genotypes that are adapted to specify environmental conditions.

Molecular markers offer a powerful tool for designation intents and in engendering programmes. Minor harvests, such as bambara Indian potato, could profit from the application of such engineerings in molecular genteelness ( Massawe et al. 2002 ) . The Deoxyribonucleic acid polymorphism detected utilizing SSRs in bambara Indian potato in this survey further opens the possibility of developing a molecular familial map that would be utile for familial sweetening of Vigna subterranean. Using molecular markers could hold general application for comparings to unequivocally separate bambara landraces which could later ensue in the word picture and the release of bambara adapted to specific agro-ecological zones to maximize output.

Primer 30 could non bring forth clear stria forms and this may be attributable to the specificity of the primer or as a consequence of the presence of a secondary substance like bubble in the sample. Choice of parents for mapping surveies could besides greatly benefit from the present survey Masawe et Al. ( 2002 ) ; nevertheless, surveies are still needed to compare consequences from different familial marker systems. This present survey could be enhanced with farther research to measure the familial diverseness within the related bambara Indian potato landraces utilizing more than one molecular technique. Albeit, our aim of placing the diverseness between the 24 landraces being achieved, our hypothesis of non-relatedness between the landraces was disproved.