Final Report “Genetic enhancement of Dolichos bean through integration of conventional breeding and molecular approaches” Sponsored by The Kirkhouse Trust, United Kingdom Project Leader Dr. M. Byre Gowda Sr. Scientist (Plant Breeding) AICRP on Pigeonpea, GKVK, Bangalore – 560065. University of Agricultural Sciences GKVK Campus, Bangalore – 560065
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Final report -1 - Kirkhouse Trust · Final Report Dolichos bean, Lablab purpureus L. Sweet (2n = 22) belongs to the family Fabaceae and is one of ... 1 Dr. G. Girish Res. Associate
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Final Report
“Genetic enhancement of Dolichos bean through integration of conventional
breeding and molecular approaches”
Sponsored by The Kirkhouse Trust,
United Kingdom
Project Leader Dr. M. Byre Gowda
Sr. Scientist (Plant Breeding) AICRP on Pigeonpea,
GKVK, Bangalore – 560065.
University of Agricultural Sciences
GKVK Campus, Bangalore – 560065
Final Report
Dolichos bean, Lablab purpureus L. Sweet (2n = 22) belongs to the family Fabaceae and is one of
the most ancient crops among cultivated plants. It is presently grown through out the tropical regions in Asia
and Africa. It is cultivated either as a pure crop or mixed with other crops, such as finger millet, groundnut,
castor, corn, bajra or sorghum in tropical region. It is a multipurpose crop grown for pulse, vegetable and
forage. The crop is grown for its green pods, while dry seeds are used in various vegetable food
preparations. It is also grown in home gardens as annual crop or on fences as perennial crop. It is one of the
major sources of protein in the diets in southern states of India. The consumer preference varies with pod
size, shape, colour and aroma (pod fragrance).
Within India, Lablab as a field crop mostly confined to the peninsular region and cultivated to a large
extent in Karnataka and adjoining districts of Tamil Nadu, Andhra Pradesh and Maharashtra. Karnataka
contributes a major share, accounting for nearly 90 per cent in terms of both area and production in the
country. Karnataka state records production of about 18,000 tonnes from an area of 85,000 hectares. Outside
India, the crop is cultivated in East Africa, with similar uses, and in Australia as a fodder crop.
The efforts of improving the crop utilizing indigenous and exotic germplasm have been useful in
breaking the yield barriers to a small extent resulting in a compact plant type, reduced duration and photo-
insensitive types. Despite the many good attributes, the crop has remained unexploited owing to low
productivity, long duration, photosensitivity and indeterminate growth habit. Hence comprehensive
germplasm collection and evaluation together with development of suitable genotypes with desirable traits
for a pure crop and investigation of its value as an inter-crop with other food and forage crops are essential.
To exploit these resources, a combination of genetic and phenotypic data is required to assist conventional
breeding programmes to achieve the targeted aims efficiently. With all this background information, the
following objectives are formulated with time-linked outcomes.
1. Collection of germplasm and phenotypic characterization of morpho-agronomic traits with emphasis
on resistance to pod borers and bruchids.
2. To develop high yielding genotypes having suitable plant type for mono and intercropping systems.
3. Molecular characterization and identification of marker/trait associations for marker assisted selection
to develop varieties for desirable traits.
4. Testing and validation of advanced breeding lines for improved traits.
I. Recruitment of staff
The following persons were worked against the sanctioned posts. The details are as follows.
Sl No
Name Job Title Salary Date of Joining
Date of leaving
1 Dr. G. Girish Res. Associate Rs.18, 000/- 01/03/2006 12-03-2009 2 Mr. Ganapathy SRF Rs. 14,000/- 01/02/2008 30-06-2008 3 Miss Nagrathnamma SRF Rs. 14,000/- 19/07/2008 31-01-2009 4 Mr. Suresha Field Assistant Rs. 5000/- 01/03/2006 31-01-2009 5 Mr. Subramanya Lab Assistant Rs 5000/- 01/09/2006 28-02-2009 6 Mr. Lingaraju Driver Rs. 5000/- 23/05/2006 28-02-2009
II. Purchase of Vehicle
A New vehicle was purchased on 3rd May 2006 for Rs. 7, 05,067.
The vehicle was used for collection programmes; conduct of multilocation
trails at different locations and in farmers’ fields, purchase of inputs and for
training programmes.
III. Construction of Seed Storage building
Opening ceremony of the seed storage building was held on 09.01.2008. Building was inaugurated
by Sir Edwin Southern and Madam Dr. Sonia Morgan and Presided by Dr. P .G. Chengappa, Vice
chancellor, UAS, Bangalore. The ceremony was attended by Dr. H. S. Sharathchandra, Emeritus Professor,
Human Genome Project, IISC, Bangalore, Dr. T. K. Prabhakara Setty, Director of Research, Dr. T. K. S.
Gowda, Director of Post graduate studies and Dr. R. Govindan, Director of Instruction (Agri) and other
dignitaries, students and staff of the university.
Two Freezers (-20) were purchased and placed in the building for storing of seeds. We are storing
seeds kept in seed boxes at – 20 temperatures.
c. Visitors during the project period
1. Sir Edwin Southern
2. Dr. Sonia Morgan
3. Dr. Robert Kobner
4. Dr. H. S. Sharathchandra
5. Dr. Noel Ellis
6. Dr. Maggie Knox
7. Dr. Brigette Mass
8. Dr. Bruce Pengelly
9. Dr. Miriam Kinyua
III. Technical Progress:
1. Collection and Maintenance of germplasm lines
Germplasm collections maintained No.1 Local collections 3742 Exotic types 113 Collections received from UAS, Dharwad 554 Kenya collections 45 Total 485
The following are the exotic germplasm lines maintained after rejuvenation.
2. Development of Descriptor list of Dolichos The descriptor list developed at Bangalore was used for cataloguing of germplasm.
During the first year of the project, characterization of germplasm was done for various
morphological characters as per the Dolichos descriptor list developed at Bangalore based on AVRDC data and local knowledge. This was also shared with Dr. Ravikumar, UAS, Dharwad and Dr. Miriam Kinyua, Kenya. The descriptor developed at Bangalore is presented below.
3. Evaluation of germplasm for qualitative and quantitative traits.
All the collected accessions during the previous years were evaluated during 2007 and 2008. For
evaluation purpose, germplasm was grouped as indeterminates, semi determinates, pendal types and wild
types and were grown separately. All the accessions were characterized with respect to qualitative and
evaluated for quantitative traits.
a. Variability for qualitative characters in the germplasm accessions
Sl. Character Type No. of Germplasm White ( 1) 80 Green ( 2) 322
1 Emerging cotyledon colour
Purple ( 3) 0 Green ( 1) 402 2. Hypocotyl colour Purple (2) 0 No pigmentation (0) 314 Localized to node (3) 47 Extensive (5) 29
3. Stem pigmentation
Almost solid ( 7) 12 Green (1) 363 4. Main colour Purple (2) 39 Absent ( 0) 402 5. Leaf anthocyanin Present ( 2) 0 Pale green (1) 171 Green (3) 213 Dark green (5) 189 Purple (7) 0
Amongst the germplasm lines evaluated over two years, GL 187 showed resistant reaction to
Helicovrapa, Adisura and Bruchids infestation.
5. Breeding for high yield with desirable traits
In order to develop high yielding genotypes, different crosses were affected involving the newly
identified variety HA 4 and germplasm lines. Different cross combinations made over the years is presented
below.
sl Cross combinations 1 HA 4 X GLB 16 2 HA 4 X GLB 91 3 HA 4 X GLB 121 4 HA 4 X GLB 124 5 HA 4 X GLB 153 6 HA 4 X GLB 122 7 HA 4 X GLB 43
During the subsequent years, different generations of segregating materials (F1 generation to
F4 generation) were raised and selections were made in the segregating populations.
F2 generation
In the F2 generation, these populations showed segregation for many traits viz., growth habit,
plant type, flowering, maturity and flower color, pod characters and seed color. We had imposed selection in
these populations for desirable traits which are having high yield, determinate growth habit, and high
number of branches, good pod fragrance and constricted pods.
Sl. Crosses No. of selections Type of selections made
1 HA 4 X GLB 16 Early types -40 Med duration-82
2 HA 4 X GLB 91 Early types –68 Med duration-18
3 HA 4 X GLB 121 Early types –6 Med duration-24
4 HA 4 X GLB 124 Early types –14 Med duration-36
5 HA 4 X GLB 153 Early types -46 Med duration-144
Growth habit, plant type, pod shape, pod constriction, pod fragrance, pod number, branch number, long pod bearing branches, seed type, high shelling percentage
6 HA 4 X GLB 122 Early types –8 Med duration-32
Growth habit, pod bearing at nodes, pod shape, pod constriction, pod fragrance, pod number, branch number, long pod bearing branches, high shelling percentage
7 HA 4 X GLB 43 Early types –16 Med duration-38
Growth habit, pod shape, pod constriction, pod fragrance, pod number, branch number, long pod bearing branches, seed type
8 Sel 246 X GLB 122 Early types –11 Med duration-31
Growth habit, pod bearing at nodes, pod constriction, pod fragrance, pod number, branch number
Crossing HA 4 GLB 122
F2 generation
F3 generation
All the early selections from the above crosses were forwarded for F3 generation. These
selections were raised in plant to progeny rows. Progeny rows exhibiting uniformity for the desirable
characters and few high yielding single plants from other rows were selected. These selections will be
forwarded to F4 family confirmation.
F4 generation
The selections made in F4 were forwarded to F5 generation. From these progenies, progenies
selections were made based on earliness, branch number, pod type, pod number, pod fragrance and growth
habit (both determinate and semi determinate types).
F5 generation
The selections made in F4 generation during the first part of the season of 2007 were sown were
grown again during March 2008 at Hebbal. Few high yielding lines were also included in the evaluation
trail. The crop is at flowering, pod maturity and harvesting stage.
F6 generation
The progeny selections made in F5 generation of the following crosses during the previous year
(2007) were sown in March 2008. From these populations, high yielding progenies (20) were selected based
on their performance on yield, duration, growth habit, pod number, pod shape, pod fragrance and named
them as HA selections.
In addition to above, we have made other crosses also and they are in different generations. Selections were made from these crosses also for desirable traits. The progress on these populations will be reported in the next years report.
Sl. Crosses No. of F5 progenies row sown
No. of selections Made in
1 HA 4 X GLB 16 10 03 2 HA 4 X GLB 91 03 01 3 HA 4 X GLB 121 08 02 4 HA 4 X GLB 124 18 03 5 HA 4 X GLB 153 18 05 6 HA 4 X GLB 43 11 03 7 HA 4 X GLB 122 13 01 8 Sel 246 X GLB 122 15 02 20
6. Evaluation of breeding material in the Multilocation trial:
All the 20 advanced breeding lines were subjected to post harvest observations for seed weight, seed
colour, seed shape and seed lustrous ness. From these 20, finally 12 lines based on desirable characters were
selected viz., HA 7-1, HA 7-2, HA 7-3, HA 7-4, HA 7-5, HA 7 -6 HA 7-7, HA 8-1, HA 8-2, HA 8-3, HA 8-
4 and HA 8-5. All these 12 lines along with check HA 4 were evaluated during September-December 2008
both at Bangalore and ARS, Balagigapade. The data is presented below. Data on yield & yield attributes of multilocation trail in Dolichos
Seed yield (Kg/ha) Sl. no.
variety GKVK Balgigapade Mean
1 HA 7-1 640 571 605 2 HA 7-2 642 590 616 3 HA 7-3 317 537 427 4 HA 7-4 508 375 442 5 HA 7-5 448 437 441 6 HA 7-6 455 520 488 7 HA 7-7 507 447 477 8 HA 8 -1 334 346 340 9 HA 8-2 349 480 415 10 HA 8 -3 425 419 422 11 HA 8-4 379 400 390 12 HA 8-5 501 466 484 13 HA 4 483 518 501
Amongst new entries tested, the line HA 7-2 with a yield potential of 616 kg/ha recorded highest
seed yield followed by HA 7-1 (605 kg/ha) compared to HA 4 (501 kg/ha). In general, the yield levels are
low due to persistent rains at the time of flowering which resulted in flower drop. Continuous rains at the
time harvest of the crop have also resulted in fungus growth on pods and which resulted in low yield.
6. Development of mapping population based on the molecular markers
To develop the mapping populations, the following crosses were carried out. In some crosses, very few
Dolichos lines were also subjected energy content analysis. Values ranged from 325.27 to
343.83 K. cal. The phosphorus content varied from 415.35 to 471.56 mg, iron content ranged from
4.85 to 6.66mg, calcium content was analyzed to be ranging from 49.78 to 61.34 mg.
Energy, phosphorous, iron and calcium content in Dolichos beans
Genotypes Energy
(Kcal) Phosphorus (mg/100g)
Iron (mg/100g)
Calcium (mg/100g)
HA-4 336.88 427.59 5.59 50.98 HA-4-1 341.63 444.25 4.85 56.35 HA 4-2 340.09 418.67 6.66 49.78 HA 7-4 340.57 437.29 5.14 57.82 HA 7-6 343.83 439.42 4.97 53.46 HA 7-7 337.41 467.64 6.48 59.54 Sel 10-1 341.20 429.53 6.11 50.06 Sel 15-1 326.15 415.35 5.08 54.75 Sel 23-1 326.97 417.24 5.42 53.59 Sel 246 329.19 469.27 6.17 52.66 Sel 321 325.27 453.50 5.83 61.34 GL 20 333.12 471.56 6.39 51.76 GL 43 329.94 457.25 5.82 56.55 GL 49 331.47 451.85 5.95 60.28
The antinutrient compositions of different genotypes were analyzed. Tannin was found to
be ranging from 579.92 to 749.29 mg. The average value of phytate and oxalates ranged from
652.35 to 748.07 mg and 316.90 mg to 470.40 mg respectively.
Anti nutritional composition of different genotypes
Genotypes Tannins (mg/100g)
Phytate (mg/100g)
Oxalates (mg/100g)
HA-4 691.23 695.98 357.30 HA-4-1 658.98 652.35 470.40 HA 4-2 709.23 736.23 387.90 HA 7-4 736.54 708.05 396.48 HA 7-6 595.02 661.23 331.90 HA 7-7 634.59 725.24 433.30 Sel 10-1 582.31 713.26 350.60 Sel 15-1 749.29 670.05 343.50 Sel 23-1 612.11 687.68 365.20 Sel 246 579.92 748.07 440.00 Sel 321 600.95 668.39 316.90 GL 43 605.92 672.54 516.00 GL 49 680.09 701.99 408.10 GL 20 588.21 691.51 392.30
The genotypes Sel 15-1, HA 4-2, GL 49 and HA7-7 had higher nutrient content with
regard to protein, iron, calcium and phosphorus respectively. The genotypes Sel 246, HA 4-1 and
Sel 321 had lower antinutrient composition for tannin, phytate and oxalate respectively.
b. Development of food products from Dolichos
Green seeds of selected two lines (GL 43 and GL 49) were given to Miss B.S. Shruthi, student of
the department Food and Nutrition, UAS, Bangalore for development of food products. Six
products were developed from these two genotypes viz., Upma, Usli, Sambar, Palya, Khichidi and
fried Khara. The products were evaluated by 5 point hedonic scale for appearance, aroma, texture,
taste and overall acceptability. All the products were accepted. From the mean scores of
organoleptic characteristics, the data that varies to a maximum of 7.5 per cent with a character
shows marginal differences through the data was found significant. Therefore, it could be either of
the genotype GL 43 or GL 49 is suitable for preparation of these products.
1.
I0. Molecular work
a. Diversity studies in Lablab purpureus:
One hundred & ninety two primer pairs (table I) received from Dr.D.J.Kim, were used to
screen the selected 10 genotypes of Lablab purpureus. (HA3, Mac-1, SRL, Rongai, Highworth,
CPI36903, CPI52544, CPI52552, CPI52535 and ILRI6536) to assess the genetic diversity within
Lablab and to gain some understanding of the transferability of gene specific markers to Lablab
purpureus. These 10 genotypes comprises both Indian material and exotic material (please refer
table 2). In this study I have used the SSR markers, intron directed primers and few primers
designed based on Lablab sequence. And also I did AFLP studies for the selected 42 Indian
genotypes of Lablab purpureus with six primer.
Table I: Primers used for screening 10 genotypes of Lablab purpureus
DNA Name Sequence forward Sequence reverse Species CA906441-1 CTTCCCCACTGATTCATTTCTGTT CACTCCCCTTTGTTCTTGATTATG P. coccineus
CA909226-1 GATCTCTTTCACGCTGGACAT CACATAGCGACATGCTAGAACACT P. coccineus
CA910489-1 AGAAAAAGGAGGGAAATGCTGCTA TTGCCTCCGCTTTCTTTATCTTTC P. coccineus
CA910598-1 CCTCCAATTCGGTTCCAGCACTTC GCCCTTGATCTTCCTCTTTTTACG P. coccineus
CA911600-1 AACTGGTTCATCGCATTACTCTTC TGGCATCACCTTGTTTGGCATTAG P. coccineus
CA911990-1 TTTCATCCCCAGCATCAGCAGTTT TGGGGGTGGGGCAGAATCA P. coccineus
CA912170-1 TGGAGAAAGCATGGCAGGATACT CCCCGGAGGCAAGATTTCAA P. coccineus
CA914165-1 GATAATTTTGACGAGGGAGCAGA GGTGGAGAACAGGAGCAAAGTATT P. coccineus
CA912542-1 TTATTGGAGTTGCCCTGTTCTTCA GCCCCGTCTGCATTCCTTTGT P. coccineus
CA906286-1 CGGGCGGGGGAGATTGT CCCAGCCCACGTAAGGTAAGAAC P. coccineus
CA910928-1 CGGAATCTGGTCGGCCTACATCTA TGATACCCCTCCGGCAACAAG P. coccineus
CA907856-1 ACACTTGAGGGAAAGCCATCA TTCCAAGCCAAAGCAATAAC P. coccineus
M80799-1 ACGGCGGGCAATGTCAA AGTTCACGTGCTCCGCTGTTAT V. unguiculata CA906298-1 TAGTTGACCGGTTATCTGTTGTTT GCGCCTACGAGCATCACTTA P. coccineus Pc_CL2167-1 CATCACGCGATAATACACGACT TCTCTGCCGAAGGTTATGGA No items found CA912634-1 GACGGAATGAAGTTGAAGGTGGAT AGATGGGCCAAGATATTTTAGACT P. coccineus
CA913150-1 CGGAGATGGTGGCGTGTTATT CTTGTGCAGCCTTGTTTTCAGC P. coccineus
CA911895-1 CATCTGCTTTTGCCCATTACG CACCAAGCCGCTCTAGCAAGTTAT P. coccineus
CA911460-1 GAGCTGCAACAAGGAATGGATG ATGAGGGCTAACAGGAAAAGAAGG P. coccineus
CA902380-1 GGCCCCCTCAGACAGAT AGATAAATTTGGGCTTGAA P. coccineus
CA907467-1 AGCCTGTCAATTCCAAACTCCACT ATCCAACTTCAGCACCCAGACAAA P. coccineus
CA907742-1 TCGGTTGAAAAAGAAAAGCAGAAG ATAGGCACTCAGGAAGGGAATGTC P. coccineus
BQ481755-1 CATGGCGAAGTAACAAAAGTGGTC ATATGCAGGATGAGGAAGATGGTT P. vulgaris Pc_CL65-1 CTCGTCTCGCCACCCAGTTCT AGTCACCGCTCTCCACCCTTCT No items found CA900698-1 TTGTTCTCAGCGCAATGTGG CTGAGCACCAAAGCCCCTGACA P. coccineus
CA900229-1 TGCCGAGTCATCCCGAACAT CTCCAAAGCTGAAGCGAACCA P. coccineus
CA914163-1 ATGGTGAATGCCTGAAACAAATAC CATGACAAATGCCTGAATAAGACT P. coccineus
CA912177-1 CTTGGTCCGCCATACTCTGAT TTTTCTTTCCTCCTCTGGTATGG P. coccineus
AJ225088-3 GGTATGATGGCCGTGAGTGGT TAAAGCTGTACAACGGCGATGAA V. unguiculata
AJ225088-5 GAACACCACCGCCACACG GCACTAGCATTAACGCAAGAAACT V. unguiculata
AF287258-3 TAGCGCCATGACATTTACCTCTTA GCTCCGTTTTCTGCTTTATCTACA V. unguiculata
AF287258-5 CATTGGGCGAAAAGAAAGGAA CGCAAAAAGAAGGAGAGCAATAGA V. unguiculata
AF287258-1 AGGGGGTGAGCTTGTTTTTG TATTGCGAAGCCTTTAGTAGACCT V. unguiculata
AF287258-1C GTTTGATGGCATACTGGGACTTG CTCTTCCTCTGTTTTGCGGTTTA V. unguiculata
X91836-5 ATCGCGAAAAATGAATTATCTGGT TTATCGTCGGTGAAAACTGCT V. unguiculata
X91836-5C CCGGAAACATGGCATTATTATTAG CCATTGCCTCGTTCCCATCTT V. unguiculata
X91836-3 AGTCTCCACCTCCCCCTTCT TTTAGCATTTTGAGGCACTGATTC V. unguiculata
AB027154-3 ATAAAATACCACAACAAAGGAGAT CTTATTGGTTGATGTAGTGGAT V. unguiculata
AB028025-3 CGTTGCGCCGCCTTTTAGTTTAC TCAATTTGGCAGGATGAACC V. unguiculata
AB030293-3 ATACGCCCCCGGAATCTAC TAGCGGAACAAAACAAGAAGAATA V. unguiculata
AB030294-3 AAGGTGGTGAGGGCGAGAAG ACCAACCAAACCACAGAAATGAC V. unguiculata
AB030295-5 TGGGCGAGCTTACAACATACACA CGGCCCCACTTCCACCTT V. unguiculata
AB030295-3 TCGCATTCGCCCATCTGAG GCTTAGCATTGCTTACTCCATTCA V. unguiculata
AB030295-1 GTGCCTGCTGACATTGAAACTG ACGGAATCCCCAAGCAAAGTC V. unguiculata
AB038691-3 GACGAAGGCTTGGCTCTCC TTACCCACAAAAGTAAATCAAAT V. unguiculata
AB038692-3 AAACATTTGGCGTTTGAAGT CAGCATATGCATGAACCAAGTAGC V. unguiculata
AB056448-3 GCTTCAATTTGCTCCTTATGG TCATTTGTTTTATTTGAAGTGTCG V. unguiculata
AB056448-1 TGTTTTGGGCCATCAGAGG CGTCCAATTTATTCGCTACAAGTT V. unguiculata
AY189137-5 TGTTATCTGGGTTTTCTCGTCTTG TGATGGCGCTAGTGATGATTTT V. unguiculata
AY189137-3 GACACCGGGCGTATCCTT CTTGCTTATTATATGTTGCCTTAG V. unguiculata
AY189137-1 TTCGTGTCTGGGGGAGGAT TACGCCCGGTGTCATAGTGTT V. unguiculata
AY189138-3 GGCTGCAAGGGTTCTCAATG CAACAATATGCCTCCTTCTGC V. unguiculata
AY193835-3 AAATGGTTGCTTTCTCTGACA GCAACATTTGTATGGGGAACCT V. unguiculata
AY193836-3 GGGCAACCAAACCGTGTG ATGGAAGCAGAAATTTGAGTAAC V. unguiculata
AY193837-3 ATATCGGCGCCTCTTCCCTACAGT GACATAAAACTCCCACGAAATCAG V. unguiculata
AY193837-1 CCATTTGTACCACCCAGGAG ATCGGCAATGACAGGAACA V. unguiculata
AY257179-3 AACTCAGGCAGATCGCTTTGT TACATGTAATTTTTCTTCAACTCT V. unguiculata
AY257179-1 AGCCAAGCCTCTGCCATTC GACAAAGCGATCTGCCTGAGTT V. unguiculata
D83970-3 TCAACCAGTATAATCGCAAGACAT CCAGCGACATCATCACAACAATAA V. unguiculata
D83971-3 GCTTGGGGCTTGAATTTACTCCT AATGCAAACTCTTACAAACCACAC V. unguiculata
D83971-1 CCAGCTTTGAAGGGGACTCT TGGGCAATTGCAACATCTCT V. unguiculata
D83972-3 AGAAAGGGATAGTGGACAAGATTA TTACATATCCATTGGCAGAACATC V. unguiculata
D88121-3 CCTAACGATGTGGCAGAAGC ATGGCTAGATTTGAGTGAGGATTG V. unguiculata
D88122-3 CGCCGGACGAGGAGTAT AAAAGAAATTGGGTTAAAAAGTAT V. unguiculata
U30875-5 TTGGGATTAAGCTTCTGATTTTGA ATTGATGGCGCTAGTGATGATT V. unguiculata
U30875-3 TTTGGAGGCAAGGGCTACTA TTGCTTATTATATGTTGCCTTAGA V. unguiculata
U30875-1 CAATTCACGAGGCCTCTAAAAAG TACGCCCGGTGTCATAGTGTT V. unguiculata
U30895-5 TGCCACCGCTTCTGCTTATCTCAC CGCCCCCGAAGTCACAATG V. unguiculata
U30895-3 GGAGGCTGGGAAGATAGAAGACT ATGCGAGATCAAGATAAGCCTAAA V. unguiculata
U30895-1 ATTGTTGCCGGTGACGTTATTATT ACTCGGGGTATGTTATCTGTGAAA V. unguiculata
U30896-3 GTCGGGATATTGGCTGGAGAG GAGATGGGGGTGGATGAATAGTT V. unguiculata
U30896-1 TCGGCAGAAGGTTCCAAGTTT GATACCCCATTCAATTCTCCTCTG V. unguiculata
U33205-3 CTTTTTGGATTGGTGCGTGAT AACAATTTGATATGGGGAACCTC V. unguiculata
U61379-3 GGGCCCATGGACCTCAAAC TAAGAACGCAAAGAACGAAACATC V. unguiculata
U61379-5 GTCCGGTGGTTCTGCTCGTGTC CTGCCCGGGTGGAAAGGAAC V. unguiculata
U61396-3 AGAACCAGCTCAGCCGAAATCAGA GGAGCGTGCTATGGGCTTATG V. unguiculata
U61396-1 TTAAACCGCAAAACAGAGGAAGAG CATCGGGCTTGGCATTTTATC V. unguiculata
U61396-5 TTCCCTCATTTCTCTTCTTTTCTC GCCGGACCTGTACTTAGCAT V. unguiculata
U85250-3 AAATAGCATTGCCAGTGAAGA GTAAGCGACTAATGATAAAGATAA V. unguiculata
U92656-5 GTCCGCGAACACGAACA CCATTTGGGATTAGTAGTTTCATT V. unguiculata
U92656-3 GGGGTTGCCAGTGAAGGAGAG TGCTAACCAACACCACCAGATAAC V. unguiculata
X74821-3 ACCAGAGATGTTTTGAGTGATTAT GTACAAGCCAAGTACTCTAACAAG V. unguiculata
X74821-5 CCATCAATAGAATCGGAGTGTT GGCTGCCCCCATTCCTTTAT V. unguiculata
X79604-3 AATTGCGGCGTTACCTACC ATAGATATGATGATTCCCTGGTGA V. unguiculata
X86030-3 CTACCCCTACCTCTATAACTCACC CAATACAAACGCATGGGCTACAC V. unguiculata
X88800-3 TGGAGTGCGGCAGAGGACA TTATTGAAAAGTGATGGAAGTTAT V. unguiculata
X88800-5 GAGCGTAGCAGTGGTGGTGTGG TCCCCCGGTTGTTTCGTGAGAC V. unguiculata
X88801-3 CCCGGCCGATGTGTTGACCTCTA CAACATAACATGCGCCTACAAT V. unguiculata
X88802-1 CTCCGCCGACGACGCAACACA AGAAGCCGGCACTCCCAAAAACAG V. unguiculata
X88803-3 CAGCTGCTTTTGCCCATTTC TCCGCGAAGAGCATTACAA V. unguiculata
X89400-3 TTTGCGCAGAATTTATGGGTTTTG TAATTTTATCAATCAACGCATCTT V. unguiculata
X89400-1C CATCGAGACTTGAAACCACAGAA CAACTGGGGTAGAATAATGACGAG V. unguiculata
X90487-3 CTGGGCTCCTTTGTGGTTTTG CACACCCTTTTGCCCTTCTC V. unguiculata
X98608-3 CCCCACCCAACGAAGATGAA AAAGATACAAAGATACCCAGAAGC V. unguiculata
Y08624-1 AATCAAGGCTTCGTTTTCATCATC GCTTGCGCTGCCTTCTCTT V. unguiculata
Z21954-3 GTCGCAGGAAACCAGAACAG TGCGAATTTAGGGCATCAA V. unguiculata
CA906441-1 CTTCCCCACTGATTCATTTCTGTT CACTCCCCTTTGTTCTTGATTATG P. coccineus CA909226-1 GATCTCTTTCACGCTGGACAT CACATAGCGACATGCTAGAACACT P. coccineus CA910489-1 AGAAAAAGGAGGGAAATGCTGCTA TTGCCTCCGCTTTCTTTATCTTTC P. coccineus CA910598-1 CCTCCAATTCGGTTCCAGCACTTC GCCCTTGATCTTCCTCTTTTTACG P. coccineus CA911600-1 AACTGGTTCATCGCATTACTCTTC TGGCATCACCTTGTTTGGCATTAG P. coccineus CA911990-1 TTTCATCCCCAGCATCAGCAGTTT TGGGGGTGGGGCAGAATCA P. coccineus CA912170-1 TGGAGAAAGCATGGCAGGATACT CCCCGGAGGCAAGATTTCAA P. coccineus CA914165-1 GATAATTTTGACGAGGGAGCAGA GGTGGAGAACAGGAGCAAAGTATT P. coccineus CA912542-1 TTATTGGAGTTGCCCTGTTCTTCA GCCCCGTCTGCATTCCTTTGT P. coccineus CA906286-1 CGGGCGGGGGAGATTGT CCCAGCCCACGTAAGGTAAGAAC P. coccineus CA910928-1 CGGAATCTGGTCGGCCTACATCTA TGATACCCCTCCGGCAACAAG P. coccineus CA907856-1 ACACTTGAGGGAAAGCCATCA TTCCAAGCCAAAGCAATAAC P. coccineus M80799-1 ACGGCGGGCAATGTCAA AGTTCACGTGCTCCGCTGTTAT V. unguiculata CA906298-1 TAGTTGACCGGTTATCTGTTGTTT GCGCCTACGAGCATCACTTA P. coccineus Pc_CL2167-1 CATCACGCGATAATACACGACT TCTCTGCCGAAGGTTATGGA CA912634-1 GACGGAATGAAGTTGAAGGTGGAT AGATGGGCCAAGATATTTTAGACT P. coccineus CA913150-1 CGGAGATGGTGGCGTGTTATT CTTGTGCAGCCTTGTTTTCAGC P. coccineus CA911895-1 CATCTGCTTTTGCCCATTACG CACCAAGCCGCTCTAGCAAGTTAT P. coccineus CA911460-1 GAGCTGCAACAAGGAATGGATG ATGAGGGCTAACAGGAAAAGAAGG P. coccineus CA902380-1 GGCCCCCTCAGACAGAT AGATAAATTTGGGCTTGAA P. coccineus CA907467-1 AGCCTGTCAATTCCAAACTCCACT ATCCAACTTCAGCACCCAGACAAA P. coccineus CA907742-1 TCGGTTGAAAAAGAAAAGCAGAAG ATAGGCACTCAGGAAGGGAATGTC P. coccineus BQ481755-1 CATGGCGAAGTAACAAAAGTGGTC ATATGCAGGATGAGGAAGATGGTT P. vulgaris Pc_CL65-1 CTCGTCTCGCCACCCAGTTCT AGTCACCGCTCTCCACCCTTCT CA900698-1 TTGTTCTCAGCGCAATGTGG CTGAGCACCAAAGCCCCTGACA P. coccineus CA900229-1 TGCCGAGTCATCCCGAACAT CTCCAAAGCTGAAGCGAACCA P. coccineus CA914163-1 ATGGTGAATGCCTGAAACAAATAC CATGACAAATGCCTGAATAAGACT P. coccineus CA912177-1 CTTGGTCCGCCATACTCTGAT TTTTCTTTCCTCCTCTGGTATGG P. coccineus AJ225088-3 GGTATGATGGCCGTGAGTGGT TAAAGCTGTACAACGGCGATGAA V. unguiculata AJ225088-5 GAACACCACCGCCACACG GCACTAGCATTAACGCAAGAAACT V. unguiculata AF287258-3 TAGCGCCATGACATTTACCTCTTA GCTCCGTTTTCTGCTTTATCTACA V. unguiculata AF287258-5 CATTGGGCGAAAAGAAAGGAA CGCAAAAAGAAGGAGAGCAATAGA V. unguiculata AF287258-1 AGGGGGTGAGCTTGTTTTTG TATTGCGAAGCCTTTAGTAGACCT V. unguiculata
AF287258-1C GTTTGATGGCATACTGGGACTTG CTCTTCCTCTGTTTTGCGGTTTA V. unguiculata X91836-5 ATCGCGAAAAATGAATTATCTGGT TTATCGTCGGTGAAAACTGCT V. unguiculata X91836-5C CCGGAAACATGGCATTATTATTAG CCATTGCCTCGTTCCCATCTT V. unguiculata X91836-3 AGTCTCCACCTCCCCCTTCT TTTAGCATTTTGAGGCACTGATTC V. unguiculata AB027154-3 ATAAAATACCACAACAAAGGAGAT CTTATTGGTTGATGTAGTGGAT V. unguiculata AB028025-3 CGTTGCGCCGCCTTTTAGTTTAC TCAATTTGGCAGGATGAACC V. unguiculata AB030293-3 ATACGCCCCCGGAATCTAC TAGCGGAACAAAACAAGAAGAATA V. unguiculata AB030294-3 AAGGTGGTGAGGGCGAGAAG ACCAACCAAACCACAGAAATGAC V. unguiculata AB030295-5 TGGGCGAGCTTACAACATACACA CGGCCCCACTTCCACCTT V. unguiculata AB030295-3 TCGCATTCGCCCATCTGAG GCTTAGCATTGCTTACTCCATTCA V. unguiculata AB030295-1 GTGCCTGCTGACATTGAAACTG ACGGAATCCCCAAGCAAAGTC V. unguiculata AB038691-3 GACGAAGGCTTGGCTCTCC TTACCCACAAAAGTAAATCAAAT V. unguiculata AB038692-3 AAACATTTGGCGTTTGAAGT CAGCATATGCATGAACCAAGTAGC V. unguiculata AB056448-3 GCTTCAATTTGCTCCTTATGG TCATTTGTTTTATTTGAAGTGTCG V. unguiculata AB056448-1 TGTTTTGGGCCATCAGAGG CGTCCAATTTATTCGCTACAAGTT V. unguiculata AF077224 AGCTGAAGCCGCCACCATA AGCAGCAGCCTTAAACTCATCAA V. unguiculata X89400 ATTGTTAGGTTGCAGGATGTAGTG TGCCACAGAGAATTTGATAGAGGA V. unguiculata AF279252 AGCTTCGCCAACTCCTCCATCAT TCATCAGGGTCAAAGCTCATCAAT V. radiata AF151961 TTCCGCCATCACCTCTTCATTCC CCGGCGACGTCAGGGTTCTTA V. radiata AF402602 CCAGGGTTCAGGTTCCATCC CCCTGTTGCATACTTACGGTCTCT P. vulgaris D13557 GAACGGATCCCAGAACGAG GGAACACCCAAATCATCAAATAA V. radiata AY189907 TGCAGTGGGAGTATCAAGTT CTGCACCATTCCAGTCACCCT P. vulgaris U85250 ACTGGGCACAATTCCTATCTGACA GGTGGCCATCTTTGCAACTTTAGC V. unguiculata BQ481569 TTTGTGTTCGTCTGAGGCGTCTTC CCCGGGTCAGTTCATGTTGCTAC P. vulgaris AB056453 ATGGGCAGGGAGCGACAGATTTAT CAGTTGCCCCGGGTGCTTCC V. unguiculata AB062360 CCCTCGGCTATAGCATTGAAGAC ACGCATAAACAAAGAGGCTGGACT P. lunatus CA901109 CCAGCCAGCCATACCCATCT CTCGACCCACATAATCTTTCAGGA P. coccineus CA901635 TAGGAGAAATGGGGTTGGCTTGAA TAAACTTGCTCGGCGTGTAATGAT P. coccineus
CA901660 AACGGGGTCCCAGATGTTGT TCTCTAGGCCTTGTTGCTCA P. coccineus
CA910054 CCGCCGGCAAGAAAGTTATCAT GGCAGCACCATCAGCAAGGAA P. coccineus
CA912710 GGTGGCTGTTGGATGTGGATG CTGTGCCGCCAAGCATTCC P. coccineus
CA909621 GAGCTTCCGTTTCATACCATTAG CTTTCCCTCGCCTTCAAATA P. coccineus
D10266 TACGAGGCATTTGGTTTGACAGTG AGCCGGTTCCTCCATTTCTT V. radiata CK151423 TTTTCATGCGACTAGTTTATCCTT AACTGGCACTCTTTCTGGTCTTA V. unguiculata CB968060 ATTTCCGAGCTTGACACATT CAAAACCACCAAGCCACATTC V. unguiculata Pc_CL338 AGCTCCAGATACTTGTTGTTGTTC ATATGGGGGTGAGGGTGACG P. coccineus
CA906101 AACACGCGGTACTACGAAATCCTC CTCCGCGTCTCTGTCTCCTACCTC P. coccineus
Pc_CL593 ATCCTGTTTCGGTGCTGCTTCTTT GGCTTCCTCCGGCGATTCC P. coccineus
AF165998 TTTTGCCCAAGGAGGTAG TTTGATTTATAAGTGGGTAGAAGA V. unguiculata U08140 GACTTTGGCCTCTCCGTTTTCTTC ATCTATATGTCCCTTCAATACTGC V. radiata U10419 TCCCACGATCTGTTTGAGC CTGCATTAGTTTTTGTTTAGATTG P. vulgaris
U28645 GGGAAGCTTGGGAGAATAGTTTTG GATTTCCCTGCTGGTTTGGTCTCT P. vulgaris
U54703 TCGGTAAGAAAAAGGAAGAAGAGA ATGAGGTGGCAGCAGGTGGTG P. vulgaris
U70531 GGCTGAATTACTACCCTCCGTGTC AGCAGCTCTTGTATCTCCCATTTG P. vulgaris
U92656 CGATGAAGATCCGCGAAACTAT TCTGGCCTCTGGAATGTGTC V. unguiculata X53603 TTGGCCCCATTAAAATAGTGACA CCTGGTTGGGTGGGGTGCTC P. vulgaris U20809 TCCGCAACAAGAGTGAACAG GACAGCAGCAATATCTACCAACC V. radiata BQ481672 ATTTTTGGTGTGCTTTCGTTTAT TCCGTGGCTTGCTGATTAG P. vulgaris CA900138 CACCGTCACCATATTCTCTGC TCTCATTAGGCCGTGCTGAC P. coccineus
CA898279 CCGTAGGATTGCTGATGAGG TTCCGGTTATGTAAAAGATGTCC P. coccineus
CA898807 TGCTGGTTTTGCGGAGGTT CCCTGTAGGCTTCCATCATCTCTT P. coccineus
CA898809 GTACCCAGTCACCAATCATAAAG AATCCCAACAACCAAGTCATCAGA P. coccineus
CA899061 GTTCTCCCCACCCTTCACATC CACGTTGCAGGCTAATAAAATAAA P. coccineus
CA899306 GAAGGGCTTTGGATGGTC TGGGAGTTACGCAAAATAGGT P. coccineus
CA899524 CTCTTCTTCAGGCTCACTCACACT TCGCGATCGGAATAACAAG P. coccineus
AY193836 TGCTACGGCCATCCAACACC AGCCAACCAACCTCCACCAAC V. unguiculata CA901208 GGGGAGCTGTCACAAGAAGAAAT TGCACATCCCCAGTCAATA P. coccineus
CA902017 ACTTCAAGGAGATGGGAGGTTATT TGTGTTATTGGTTGAGGCAGGAG P. coccineus
CA902145 TTGATGACTTCCACCGGTTCTTA ATTTCGCTATGGAGGCTTCAA P. coccineus
CA902154 ACAAGGCTGATTTAGGTTCTCC TGTATCAGCGCGTCTTTCAT P. coccineus
CA906247 ATCTGGGCAAGCAAAGAGGT AGAAGTTCCCATTCGTAAGTTGTT P. coccineus
CA906300 TATTAGAGGGATTGAGTGGGATGC CAAGCTTCAAATATACTTCTTCAGG P. coccineus
BQ481774 GACGTCGTTGTGCTCTCG GTTGGGTACCCAAAAACTCG P. vulgaris Table 2: Lablab purpureus accessions used for screening the gene specific markers
Sl.No. Lablab accessions Country of origin 1 HA3 India 2 Mac-1 India 3 SRL India 4 Rongai Kenya 5 Highworth Ex.India 6 CPI36903 Ex. Ukrane 7 CPI52544 India 8 CPI52552 India 9 CPI52535 India 10 ILRI6536 Ex. Ethiopia
II. Phenotypic and molecular characterization of germplasm
The 224 accessions collected and maintained in the KT funded Dolichos were used for
screening with the selected primers. For screening of these 224 genotypes we have selected 13
primer pairs (Table 3) to see the genetic diversity within Lablab purpureus These 13 primers were
selected based on the research article we published in the journal of ‘Plant Genetic Resources:
Characterization & Utilization’. The criteria used for the selection of primer was PCR
amplification pattern like presence / absence of bands, banding patterns and polymorphism on
1.5% agarose gel among the 10 genotypes of Lablab purpureus (HA3, Mac-1, SRL, Rongai,
Highworth, CPI36903, CPI52544, CPI52552, CPI52535 and ILRI6536).
Table 3: Primers used to screen the 224 germplasm
Sl no. Sequence ID Species of origin Sequence information Sequence class 1 AF 067417 Lablab purpureus fril cDNA EST derived* 2 AY 583516 Lablab purpureus 5S rDNA Nuclear DNA: rDNA repeat3 AB 176567 Lablab purpureus Met 2 cDNA EST derived* 4 AY 049047 Lablab purpureus pDLT cDNA EST derived* 5 AY 189907-1 Phaseolus vulgaris Genic Genomic DNA** 6 BQ 481672 Phaseolus vulgaris Genic cDNA EST derived* 7 AY 193836-1 Vigna unguiculata Genic cDNA EST derived* 8 AF 327406-1 Vigna unguiculata Genic cDNA EST derived* 9 AJ 225088-3 Vigna unguiculata Genic cDNA EST derived* 10 AW 256794 Medicago truncatula TC(17) cDNA EST derived*** 11 AW 684360 Medicago truncatula CGA(8) cDNA EST derived*** 12 AW 127626 Medicago truncatula GTTT(7) cDNA EST derived*** 13 AW 186493 Glycine max CTT(13) cDNA EST derived*** *: aiming for intron spanning from genomic DNA template, **: gene where the exon & intron positions are known, primers amplify across an intron, ***: containing an SSR portion that is spanned by the primer
Molecular marker development
We have sequenced gene fragments from nine Lablab genotypes GL48, GL147, GL153,
HA-4, CPI24973, CPI31113, Highworth & Rongai (for the origin of these Lablab accession please
refer table VII) along with a sample from cowpea genotype C-152 corresponding to fril (mannose
products (5 µl of each) were visualized on 2% agarose for amplification. Then 1µl of PCR
amplified product is used as template DNA, 1µl of Big dye, 1 µl of sequence buffer, 1µl of 3.2 µM
primer and the PCR conditions were as follows: 960 C/1 min; (960 C/10 s: 560 C/5 s; 600 C/4 min)
repeat for 24 cycles: 100 C 10 minutes to amplify for the sequencing experiment. The PCR
amplified DNA was sequenced using DNA analyzer from ABI. I have sequenced gene fragments
5S rDNA of amplicon size 300 base pair, fril of amplicon size 300 base pair, pDLL of amplicon
size 200 base pair, pDLT 200 base pair of amplicon size, AF287258 of amplicon size 400 base
pair, AF151961 of amplicon size 400 base pair, D13557 of amplicon size 800 base pair,
metallothionine genes Met1 of amplicon size 1000 base pair & Met2 of amplicon size 800 base
pair from the above nine Lablab genotypes plus one cowpea genotype
These DNA sequencing results showed single nucleotide polymorphism among the exotic
material in particular with CPI24973 being noticeably distinct; the Indian materials are identical
except in one case i.e. HA4 (table 6).
Table 6: DNA Sequence polymorphism among the Lablab accessions Met 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 CPI24973 C C T C C C G A A T T A C G T G G C A CPI31113 C T T C C T A A A C T A C A - - G T T Rongai T C - - C C G A T T A G T A - - A C A Highworth T C - - G C G G A T A G C A - - G C A HA-4 T C - - C C G A T T A G T A - - A C A GL48 T C - - C C G A T T A G T A - - A C A GL147 T C - - C C G A T T A G T A - - A C A GL153 T C - - C C G A T T A G T A - - A C A Met 2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CPI24973 T C T TTG G TT A T C G G - G A TCTG C A G T G CPI31113 T C T TTG G TT A T T A G - T G ---- C T A T T CPI60126 C G C --- - -- T A T A T G T A TCTG G T A T T Rongai T C T TTG G TT A T T A G - G A TCTG G A G A T Highworth T C T TTG G TT A T T A G - G A TCTG G A G A T HA-4 T C T TTG G TT A T T A G - G A TCTG G A G A T GL48 T C T TTG G TT A T T A G - G A TCTG G A G A T GL147 T C T TTG G TT A T T A G - G A TCTG G A G A T GL153 T C T TTG G TT A T T A G - G A TCTG G A G A T
D13557 AF287258 1 2 3 4 CPI24973 T T ------------- A CPI31113 C A AGCTCAACGTATA GRongai C A AGCTCAACGTATA A HA-4 C A AGCTCAACGTATA A GL48 C A AGCTCAACGTATA A GL147 C A AGCTCAACGTATA A GL153 C A AGCTCAACGTATA A AF151961 5S rRNA 1 2 CPI24973 C A CPI31113 T A CPI60126 C A Rongai C A Highworth C A HA-4 C G GL48 C A GL147 C A GL153 C A
The molecular diversity found among the Indian materials and the African materials can be
seen in Table IX. Based on the Met2 genic sequencing data at the end of my trip in JIC 2007 I
designed a pair of allele specific primers (Soleimani et al, 2003) to distinguish between CPI 24973
& the Indian genotypes by making use of single nucleotide polymorphism. When we amplify with
these allele specific primers the alleles were not distinguished as expected. Here we have used two
general primers and an allele specific primer in a PCR reaction (Soleimani et al, 2003). It was
expected a common band in both CPI 24973 and HA 4 and a lower band in one genotype
depending on the allele specific primer. But when it was amplified with HA4 allele specific primer
I got a common band and a lower band in both the genotypes, where as in case of CPI 24973 allele
specific primer I got a lower band in CPI 24973 as expected but I did not get the common band in
both the genotypes.
1 2 3 CPI24973 A T A CPI31113 T C G CPI60126 T C G Rongai T T A Highworth T T A HA-4 T T A GL48 T T A GL147 T T A GL153 T T A
1 2 3 4 CPI24973 - T G A Rongai C T G T Highworth C G A T HA-4 C T G T GL147 C T G T GL153 C T G T
Table 7: Molecular diversity within the selected Lablab accessions CPI24973 CPI31113 Rongai HA-4 GL48 GL147 GL153
CPI24973 0.00 0.68 0.42 0.46 0.42 0.42 0.42
CPI31113 0.68 0.00 0.56 0.59 0.56 0.56 0.56
Rongai 0.42 0.56 0.00 0.02 0.00 0.00 0.00
HA-4 0.46 0.59 0.02 0.00 0.02 0.02 0.02
GL48 0.42 0.56 0.00 0.02 0.00 0.00 0.00
GL147 0.42 0.56 0.00 0.02 0.00 0.00 0.00
GL153 0.42 0.56 0.00 0.02 0.00 0.00 0.00
III. Generation of mapping populations for constructing genetic map
a. Selection of distant genotypes for wide crosses based on molecular and phenotypic data
To construct a genetic map we need distant/ wide parents for making crosses to develop the
F2 mapping populations. If there is variability among the parents we are going to get variation
within the segregating populations (i.e. in F2 mapping population), if there is no variability among
the parents we can not expect variation within the segregating populations. Therefore selection of
distant genotypes for crosses is very much important in generating the mapping populations for
genetic map construction.
Based on the phenotypic data and genotypic data we have selected three genotypes GL 153,
GL 48& GL 147 in addition to HA-4 which is a released variety that is photo insensitive,
determinate, has early maturity and high fragrance. Morphological traits considered for selection
are Growth habit, Photo-sensitivity, crop duration, Pod fragrance and the 100 seed dry weight
(Table 4). These four parents were used as female parents and crossed with the CPI 24973, CPI
31113 and (this CPI material was brought from JIC during our 2006 visit). Therefore we made 12
crosses using these 7 parents (Table 5).
Table 4: Important phenotypic traits of parents used for crossing
Name of the genotypes Phenotypic characters GL 48 GL 147 GL 153 HA 4 CPI 24973 CPI 31113 CPI
60216 Growth habit Indeterminate Determinate Indeterminate Photo sensitivity Sensitive Insensitive Sensitive Crop duration Long (Annual) Short Long (Perennial) Pod fragrance Medium High Nil 100 seed weight (dry)
15.2g 14.5g 18.5g 19.8g 6.85g 6.93g 6.9g
Table 5: Details of the crosses attempted
♀/♂ CPI24973 CPI31113
GL48 X X
GL147 X X
GL153 X X
HA-4 X X
b. Development of F2 populations for genetic map construction
The F1 seeds along with their parents were germinated in water cups (4cm in diameter &
10cm in height) in the greenhouse. 30 days after sowing the seedlings were planted in the field. I
have selected the true F1 crosses by observing the phenotypes in the field, those which resemble
the female parents (due to selfing) were rejected and the other types were retained. There were
only 3 true F1 crosses and the successful F1 crosses are GL 153 X CPI 31113, HA-4 X CPI 31113
& HA-4 X as highlighted in Table VI. (The failure in crossing may be due to the inappropriate
selection of the female flower, that the female flower may be selected after the anther dehices since
the F1 plants were resembling the female parent). The phenotypic observations were recorded till
harvest. At the onset of flower initiation the whole plot was covered with net to avoid the possible
cross pollination. i.e. the F1 plants were allowed to self. DNA was extracted from the F1 plants and
the F2 seeds were collected and preserved.
Molecular marker development (Work done in JIC during Aug-Sept 2007) (contd.)
Work done in JIC during June-July 2008
We have designed 32 primer pairs based on the information available in cowpea genomic
database (http://cowpeagenomics.med.virginia.edu/CGKB) and received 26 primer pairs from
Dr.Mike Timko. We used all 58 primer pairs (details are in table X) to amplify products from nine