PertanikaJ. Trop. Agric. Sci. 20(1): 75-82(1997) ISSN: 0126-6128© Universiti Putra Malaysia Press

Analysis of Genetic Linkage in the Cowpea Vigna unguiculata

O.B. KEHINDE,1 G.O. MYERS and I. FAWOLE2

1 Present Address: University of AgriculturePM.B 2240, Abeokuta, Nigeria

department of Agricultural BiologyUniversity of Ibadan

Ibadan, Nigeria

Keywords: linkage group, genetic map, locus, character

ABSTRAKAnalisis genetik 12 lod yang mensyaratkan dri-dri morfologi menunjukkan beberapa rangkaian dalam kacangduduk (Vigna unguiculata (L) Walp) Ddengan menggunakan kedua-dua kacuk balik dan data segregasicantuman F2, 12 lod ini diletakkan pada lima kumpulan berangkai. Pg lod untuk pempigmeman nod, Pf untukbunga ungu, Pc untuk salut biji benih lidn, Na yang bermata kedl dan Br untuk salut biji benih coklat yangmembentuk kumpulan berangkat 1 dengan susunan mun^dn PgNaBrPc. Lokus Bpd untuk mencabangkanpedunkel dirangkaikan kepada Bp untuk pod kering coklat dan Dhp untuk pod yang terbuka bersam susunanmungfan Bpd Bp Dhp. Kumpulan berangkai yang ketiga mengandungi lod Crl untuk daun sesil. Bentuk daunhastate. Ha dan bilangan daun berseptrum, Spt, masing-masing bersekutu dengan kumpulan berangkai keempatdan kelima.

ABSTRACTGenetic analysis of 12 lod conditioning morphological characteristics indicated several linkages in cowpea Vignaunguiculata (L.) Walp). Using both backcross and F2 joint segregation data, these 12 lod were assigned to fivelinkage groups. The lod Pgfor nodal pigmentation, Pf for purple flower, Pc for smooth seed coat, Na for narroweye, and Br for brown seed coat make up linkage group 1 with the probable order Pg-Na-BrP-cPf The Bpd locusfor branching peduncle was linked to Bp for brown dry pod and Dhp for pod dehiscence with the probable orderBpd~Bp-Dhp. The third linkage group consisted of lod Crl for crinkled leaf and Pt for sessile leaf Hastate leafshape, Ha, and septafoliolate leaf number, Spt, belong to the fourth and fifth linkage groups, respectively.

INTRODUCTIONCowpea (Vigna unguiculata (L.) Walp)(Leguminosae) is an important grain legumecrop in tropical and subtropical regions of theworld. The genus Vigna contains about 170 spe-cies with growth habits ranging from erect, semi-upright to prostate and twining forms (olackhurstand Miller 1980).

The International Institute of Tropical Agri-culture (IITA), Ibadan, Nigeria and the GeneticsUnit of the Department of Agricultural Biology,University of Ibadan, Ibadan, Nigeria maintainmore than 12,000 accessions of Vigna unguiculata',data are recorded for more than 50 characters,while some of the lines carry mutant traits.

Cowpeas are diploid species, 2n = 22 (Faris1964; Mukherjee 1965; Pignone et al 1990) with

little genetic, and no chromosomal, divergenceof the cultivated subspecies from their wild pro-genitors (Faris 1965). Several mutations havecaused morphological and reproductive changesin the cowpea and these have led to the expres-sion of such traits as leaf, branching, non-branch-ing plants, etc. and an increase in useful geneticvariability. A recessive mutant is of value to plantbreeders as a genetic marker in isolating acci-dental selfs during hybridization and in identify-ing and evaluating marker-linked genes affect-ing specific quantitative traits. A marker mayalso be of value in surveying genetic variabilityin populations and interpopulation variability.

Reports of the inheritance of many mor-phological and disease resistance characteristicsof cowpea were reviewed by Fery (1985), The

O.B. KEHINDE, G.O. MYERS AND I. FAWOLE

following mutant traits: branching peduncle, non-petiolate leaf, crinkled leaf and septafoliolateleaf number were described by Fawole (1988)and Fawole and Afolabi (1983). They reportedeach to be under single gene inheritance withthe non-branching peduncle dominant to thebranching peduncle, petiolate leaf dominant tonon-petiolate leaf, non-crinkled leaf dominantto the crinkled leaf and trifoliolate leaf numberdominant to the septafoliolate leaf number.Monogenic segregation pattern was also reportedfor nodal pigmentation (Harland 1919), hastateleaf shape (Ojomo 1977), flower colour (Kolhe1970; (Hanchimal and Goud 1978), dehiscentpod (Aliboh et al 1995), narrow eye pattern(Spillman 1913), dry pod colour (Saunder 1960),brown seed coat colour (Spillman 1913) andsmooth seed coat (Rajendra et al 1979).

According to these reports, nodal pigmen-tation was dominant to non-pigmentation,hastate leaf shape was dominant to subgloboseleaf shape, purple flower was dominant to whiteflower, shattering of dry pod was dominant tonon-shattering, and narrow eye pattern was re-cessive to the solid eye pattern. Also, the browncolour of the dry pod was dominant to thewhite seed coat colour, while the smooth seedcoat was dominant to the rough seed coattexture. The symbols assigned to the genescontrolling each of these characters are pre-sented in Table 1.

However, there are few studies on genelinkage in cowpea and most links need furtherstudy and verification (Fery 1985). Calub (1968)reported that seed eye pattern and seed coatwere inherited independently and Harland(1919) reported close linkage for seed charac-teristics such as colour and eye pattern. How-ever, Saunders (1960) later reported that multi-ple effect of particular genes were responsiblefor the association between these traits.

Prior to the initiation of this study, no pub-lished work gave a precise linkage map of cowpeawhich could serve as a basis for further study.The lack of an encouraging foundation of goodquality marker genes probably discouraged in-terest in mapping genes in cowpea. Also, manyof the genes are involved in complex epistaticrelationships, which makes them difficult to clas-sify in segregating populations. However, theavailability of a fairly detailed gene map in cowpeawould be useful in genetic, cytogenetic and plantbreeding studies.

The purpose of this study was to investigatethe linkage relations of 12 easily scored, simplyinherited traits to facilitate selection proceduresand to initiate the construction of a genetic mapfor cowpea.

MATERIALS AND METHODS

The genetic materials were obtained from thecowpea genetics programme of the University ofIbadan, Nigeria and IITA. Preliminary screeningof all genetic stocks was carried out for the 12characters studied (Table 1). Each line was sownin 20 plastic pots with two plants per pot. Theplants were maintained to maturity, and no seg-regation was observed for these traits. Theirseeds collected for use in hybridization.

Ten plants from each parent were grownin the greenhouse and they were combined inthe following six crosses: IBPC x IT82E - 9;IBPC x TVU1; IBPC x IBS876; IfeBPC xTVU4578; R10028 x G2497; IBPC x WCIBADAN- 10. A portion of the Fl seed was sown in thefield in the first season of 1991 to produce F2seeds. All mature pods that developed on theFl plants were harvested and seeds were bulkedafter threshing and cleaning. Seeds of parentsFl, BC1, BC2, and F2 progenies from the sixcrosses were planted in the dry season (Decem-ber) of 1991 at the IITA experimental farm.The field was irrigated once every week for an8-hour period throughout the growing period.The experimental design was a randomizedcomplete block with four replications. For across, each parent had a total of 4 rows, Fl had4 rows, backcross to parent 1 (BC1) had 8 rows,backcross to parent 2 (BC2), the recessive par-ent, had 16 rows, while F2 had a total of 60rows. The length of each row was 10 m. Withinrows, the spacing was 50 cm, and 75 cm be-tween rows. Each plant was given a replicationnumber, a row number and a within-rownumber. This enabled data collection fromgermination to weighing of seed after harvestfor each plant without a mix-up.

During data collection, the characters werevisually scored into alternate classes. The F2and backcross data were analysed using the chi-square method to test the goodnessof-fit ofobserved ratios to expected ratios for eachcharacter.

Data were pooled when the chi-square testfor heterogeneity indicated that families werehomogeneous. For all categories of two-factor

76 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 1, 1997

TABLE 1Gene designation and phenotypic description of cowpea

33

Classes Line or Cultivar

Trait Dominant Recessive IBPC FE BPC IT82E-9 TVU4578 IBS876 R10028 G2497 WCIB-10

2p8o

o

8-4

Nodal pigmentation (Pg)

Branching peduncle (Bpd)Crinkled leaf (Crl)Non-petiolate leaf (Pt)

Hastate leaf shape (Ha)Flower colour (Ef)Narrow eye pattern (Na)Brown seed coatColour (Br)Brown dry pod (Bp)Seed coat texture (Pc)Dehiscent pod (Dhp)

Leaf number (Spt)

Pigmented

NormalNormalPetiolate

HastatePurpleSolidBrown

BrownSmoothDehiscent

Trifoliolate

Non-pigmentedBranchingCrinkledNon-petiolateNormalWhiteNarrowWhite

StrawRoughNon-dehiscentSeptafoliolate

* Not applicable

O.B. KEHINDE, G.O. MYERS AND I. FAWOLE

joint segregation ratios, contingency chi-squarewas used to detect linkage.

This was done by using Linkage-1, a com-puter program (Suiter et al 1983). If the calcu-lated chi-square is greater than the tabulatedvalue, then linkage is indicated between thetwo traits. After detecting linkage by test ofindependence, linkage intensities were calcu-lated using both F2 and backcross data. Link-age-1 was used to calculate recombination frac-tions and map distances, which were used todetermine the gene order.

RESULTS

The 12 loci tested showed good agreement withexpected 3:1 and 1:1 segregation ratios in the F2and backcross generations respectively. Linkagetests indicated linkage between some of theseloci and independence between others. Of the45 F2 and backcross linkage tests between genepairs in this study, 22 suggested independencewhile 23 indicated linkage (Table 2). The lociPg, Br, Na, Pc and Pf make up linkage 1. Two of

these loci, Br and Na, were linked with norecombination while they both showed tightlinkage to Pc locus for smooth seed coat. link-age group 2 is comprised of loci Bpd, Bp andDhp. The loci Crl and Pt make up linkage group3. The locus Ha showed independence from thesix loci tested against it, hence it belongs to aseparate linkage group. Also, locus Spt forseptafoliolate leaf number was not linked to anyof the loci tested against it and it thus formslinkage group 5. The respective gene orders andmap distances of these linkage groups are pre-sented in Fig. 1.

DISCUSSIONThe results of inheritance studies were in agree-ment with the references, indicating single geneinheritance of these characters. From results oflinkage tests, a relatively high incidence of link-age was observed. Five linkage groups, corre-sponding to five chromosomes, out of the 11linkage groups possible in cowpea, were estab-lished from this study. Loci Na (narrow eye) and

Linkage group 1.

P9

13.93

13.42

1.90

Ate

Br

16.13

2.33

Pc Pf

36.45

Linkage group 2. 25.97

Bpd

I

27.73

Bp38.40

Dhp

Linkage group 3. j i§d§ |

Crl Pt

Linkage group 4. Ha

Linkage group 5. Spt

Fig 1. Cowpea gene order and map distances from F2 population

78 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 1, 1997

TABLE 2Allelic constitution, F2 and backcross progency distribution, calculated recombination

values and map distances of linked traits in cowpea

I

0y

in

Alleles

Pg pg Bpd bpd

Pg pg Pf pf

Bpd bpd Pf pf

Bpd bpd Bp bp

Bpd bpd Dhp dhp

Bpd bpd Pc pc

Bp bp Dhp dhp

Bp bp Pc pc

Dhp dhp Pc pcHa ha Pt ptHa ha Bpd bpd

Pt pt Bpd bpdp g Pg p t PtPg pg Crl crlPg pg Spt sptPg pg Br brPg pg Pc pcPg pg Na naPt pt Crl crlPt pt Br brPt pt Pc pc

Pt pt Na na

Generation

F2BCF2

BCF2

BCF2

BC

F2BCF2

BCF2

BCF2

BCF2

F2F

2BCF2

F2

F2

F2

F2

F2

F2

F2

F2

F2BCF2

BC

a

55899

60117151887

65095

58399

61279

61872

57278

56134834175

352148518548491511502497553556

30557

31

Numberb

115397

2030

18276

12624

19746

15140

11264

14933

14914414381

13057820417623022022625519719332

18831

of Plantsc

20096

00

1368679168514

133508628

14551

15114713572

12176

194186

422723121617517431

17937

d

398768

1493671

120120110966386

1599193939839498266

1725864

238546

3840313925

Total

950379872320872320975255975255259255975255959255959678668310669974974974963963963974963963124963124

X2 linkage*

3.000.14

163.55*320.00*

1.890.06

163.02*119.62*70.18*78.21*13.73*22.28*

224.46*23.03*32.14*30.46*30.45*

4.72*0.550.054.50*

10.99*2.610.17

90.12*76.78*84.13*85.0*

6.38*4.54*0.03*5.05*1.17

Recombination% and

45.2349.0831.12

46.0849.3825.0715.6934.3823.5340.61

±±±

±±±±±±±

35.29g±22.2636.0837.7732.9435.7143.9448.3149.3544.2242.5146.1949.0413.1115.5913.5815.8843.1544.3249.1944.0245.16

±±±±±±±±±±±±±±±±±±±±±

SE

2.562.573.02

2.642.792.962.272.782.652.662.993.013.002.732.942.563.072.952.843.082.592.502.433.153.133.153.102.592.564.492.574.47

Map Distance (MU)and SE

74.85116.9836.45

79.98126.9227.5616.2442.1725.5456.6743.9423.9345.5549.2739.5337.1368.52

101.58125.7469.7862.8480.72

115.9113.4216.1313.9316.4565.2570.24

120.1968.8874.47

±±±

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

2.562.573.02>502.652.802.972.282.792.662.663.003.023.012.732.952.563.072.9

2.843.082.602.512.433.163.133.153.112.602.574.502.574.48

LinkagePhase

CCCCCCCCCC

ccccccccccccccccccccc

s

TABLE 2 (contd)

I

>p

8

Alleles

Crl crl Spt sptSpt spt Na NaCrl crl Na NaCrl crl Br brSpt spt Br brSp spt Pc pcBr br Pc pc

Br br Na na

Pc pc Na na

Generation aNumber of Plants

b cRecombination Map Distance (MU) Linkage

Total X2 linkage* % and SE and SE Phase

F2F2F2F2F2F2F,BCF2BCF2BC

549547551534544551716567735671956

1621821661711831749000120

1851641881941801801300060

787058645658225682306822668

974963963963963963963124963124963124

4.88*2.240.020.030.200.20

847.87*124.00*965.00*124.00*868.15*124.00*

45.0746.5549.7149.6248.9048.222.33

1.90

±±±±±±±

±

2.532.512.422.432.442.433.22

3.82

73.9883.29146.00139.22112.47110.78

2.33

1.90

±±±±±±±

±

2.532.512.452.432.442.443.22

.23

*P< 0.05a • dominant at both loci A-B-b - dominant at the first locus, recessive at the second locus A-bbc = recessive at the first locus, dominant at the second locus aaB-d = recessive at both loci aabb.

CRRRRRCCCCCC

OWO

b

i

ANALYSIS OF GENETIC LINKAGE IN THE COWPEA VIGNA UNGUICULATA

Br (brown seed coat) were the only very closelylinked loci. No recombinant was obtained fromthem. Nearness of these loci to the centromeremay account for the difficulty in obtainingrecombinants.

Gene order was consistent in differentpairwise combinations to support the linkagemap proposed. Calub (1968) reported thatcowpea seed eye and seed coat colour wereinherited independently. However, Harland(1919) had earlier reported tight linkage be-tween seed characteristics such as colour andeye pattern, and flower colour, though suchlinkages in this study are not as tight as thosereported by him.

Saunders (1960) observed that multiple ef-fects of particular genes are responsible for theassociation between seed characteristics such ascolour and eye pattern and flower colour, con-trary to the findings of this study. This is becauseinheritance studies indicated different modelsfor the characters and also because recombinantswere obtained in linkage tests.

In cases where tight linkages occurred andor no recombinants were obtained for either orboth recombinant classes, it was observed thatthis involved pigmentation genes. This could bebecause absence of pigments in vegetative partsusually leads to absence of pigmentation in otherplant parts such as flower, pod and even seed. Onthe other hand, a plant with purple pigments invegetative parts may or may not produce flowersor pods with pigments. Fery (1985) and Frahm-Levilveld (1965) have proposed that a generalcolour factor, C, must be present before any ofseveral pigment genes can be expressed.

When it is lacking the plant lacks pigmentin both vegetative and reproductive parts. Theextent to which this gene causes anthocyaninpigment to be produced varies, and so is thelocation of the pigment.

CONCLUSIONThis study has established five linkage groupscorresponding to five chromosomes from the 11linkage groups available in cowpea. These link-age groups comprise 12 loci. This is a significantcontribution towards the development of a con-cise and comprehensive cowpea linkage map.

REFERENCES

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Inheritance of three traits in crosses between

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BLACKHURST, H.Y andJ.C. MILLER. 1990. In CowpeaHybridization of Crop Plants, pg. 327-337.

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FARIS, D.G. 1965. The origin and evolution of thecultivated forms of Vigna sinensis. CanadianJournal of Genetics and Cytology 7: 433-452.

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HANCHIMAL, R.R. andJ.V. GOUD. 1978. Inheritancein Vigna. Indian Journal of Genetics and PlantBreeding 38(3): 339-342.

HARLAND, S.C. 1919. Inheritance of certain charac-teristics in cowpea (Vigna sinensis). JournalGenetics 8: 101-103.

KOLHE, A.K. 1970. Genetic studies in Vigna sp.Poona Agricultural College Magazine 59: 126-137.

MUKHERJEE, P. 1968. Pachytene analysis in Vignachromosomes morphology in Vigna sinensis(cultivated). Science and Culture 34: 252-253.

OJOMO, O.A. 1977. Morphology and genetics of twogene markers "swollen stem base** and "hastateleaf in cowpea, Vigna unguiculata (L) Walp.Journal of Agricultural Science Cambridge 88(1):227-231.

PIGNOME, D., S. CIFARELLI and P. PERRINO. 1990.

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82 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 1,1997