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. 2025 May 28;45(6):52. doi: 10.1007/s11032-025-01570-4

Marker assisted introgression of bacterial leaf blight and cowpea mosaic virus resistance into an elite cowpea cultivar C-152

Hirenallur Chandappa Lohithaswa 1,✉, Hebse Bhojappa Dinesh 2, Sidramappa Channappa Talekar 2, Kannalli Paramashivaiah Viswanatha 2, Mallana Goudra Mallikarjuna 3, Muntagodu Shreekanth Sowmya 1, Thaggihalli Veeranna Krishna 4, Poonam Singh 2, Dasannanamalige Siddeshi Ambika 2
PMCID: PMC12119430  PMID: 40453530

Abstract

Cowpea is an important grain legume crop and a source of vegetarian protein. C-152, a popular and widely adapted variety of cowpea, became susceptible to major cowpea diseases, viz., bacterial leaf blight (BLB) and cowpea mosaic virus (CpMV). Thus, in the current investigation, we have introgressed BLB and CpMV resistance from V-16 and V-57817, respectively, to the C-152 variety. A marker assisted simultaneous and stepwise backcross breeding scheme was used to recover an improved version of C-152 with resistance to BLB and CpMV diseases. Foreground (CISP markers VuMt401 and VuMt397 for BLB and SSR markers MA15 and MA80 for CpMV) and background selections were practiced using gene-specific and recurrent genome specific (72 markers) polymorphic markers. Two independent BC2F4 lines from each cross possessing blb-1 and cowpea mosaic resistance gene with maximum genome recovery of the C-152 were inter-crossed to derive an inter-cross (IC) F4 population. Among the 10 promising ICF4 progenies, the line MC 17−2 (KBC-12), showing high yielding with resistance to BLB and CpMV, was selected. The superiority of the cowpea line MC 17−2 was evident in terms of a yield advantage of 8.68 to 28.68%, 9.30–47.00%, 1.10–8.10% over different check varieties in the initial varietal trial, advanced varietal trial (AVT)-I, and AVT-II, respectively. Further, the multi-location evaluation of KBC-12 (MC 17−2) with the check KBC-9 covering zones 5 and 6 of Karnataka reconfirmed the high-yielding potential and stability of KBC-12 across tested environments, as evident from AMMI and GGE biplots. Thus, the promising cowpea line KBC-12 was released for commercial cultivation in zones 5 and 6 in southern India during 2024 and can also be used as a donor (IC652010) of BLB and CpMV resistance. Our current study is one such examples that revealed the power of marker-assisted selection to deliver improved cultivars from lab to farmers’ field.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11032-025-01570-4.

Keywords: Cowpea, Bacterial leaf blight (BLB), Cowpea mosaic virus (CpMV), Marker assisted simultaneous and stepwise backcross breeding scheme

Introduction

Cowpea (Vigna unguiculata (L.) Walp.), also known as black-eyed pea, is the most ancient crop known to man (Osipitan et al. 2021) and it is a widely adapted multipurpose grain legume in tropical and subtropical countries, including the arid and semiarid tropics (Singh et al. 1997). It is a versatile, warm-season, annual grain legume that tolerates drought and does well on wide range of soils. Cowpea belongs to the family Fabaceae, and genus Vigna. All cultivated cowpeas are considered to be members of the Vigna unguiculata subspecies unguiculata, which is native to Africa (Singh 1997). Cowpea is grown mostly for its seeds, but it is also used as a vegetable, where its tender leaves, young pods and grains are consumed. Cowpea haulm serves as a vital source of livestock feedstuff that provides cattle feed, forage, hay and silage and hence, farmers receive sizeable income from this crop (Alemu et al. 2016). It contributes to soil fertility improvement through symbiotic nitrogen fixation and prevents soil erosion by dense foliage which covers the soil surfaces (Kebede and Bekeko 2020). Further, it is a prime source of protein in the diet of a large number of people in the developing world. Owing to its nutritional benefits, it is popularly known as the poor man’s meat. In many tropical diets, cowpea grain is a key source of the essential amino acids, carbohydrates, minerals (such as calcium and iron), vitamins (B6 and B12) and protein (high in lysine and tryptophan) (Bressani 1985).

Globally, cowpea is grown in an area of 15.41 million hectares with an annual production of 9.78 million tonnes and an average productivity of 634.6 kg per hectare (FAOSTAT 2023). India stands as a major stakeholder in the world’s cowpea production, with an average productivity of 683 kg/ha. Cowpea is majorly grown in the southern and eastern parts of India, and Karnataka contributes nearly 10% of cowpea production to the nation’s food basket. However, the average productivity in Karnataka (440 kg/ha) is very low compared to the national and global average productivity (Project Coordinator’s Report 2023−24). One of the possible reasons for the lower yield in cowpea production could be attributed to the sensitivity of the existing cowpea cultivars to biotic and abiotic production constraints. Among various diseases that damage the crop, bacterial leaf blight (BLB) and viral diseases are important throughout Asia, Africa, and Latin America (Thottappilly and Rossel 1992). Bacterial leaf blight caused by Xanthomonas axonopodis pv. vignicola (Xav) (Vauterin et al. 1995) is widespread in all major cowpea-growing regions of the world (Gitaitis 1983; Emechebe and Florini 1997), causing significant yield losses of up to 60–65% (Sikirou and Wydra 2004). Even the crop may be completely damaged by BLB when vulnerable cultivars are grown (Emechebe and Shoyinka 1985). The symptoms of BLB include the formation of tiny, water-soaked, translucent spots, which are more clearly visible from the abaxial surface of the leaves (Williams 1975). These spots further expand, fuse, and develop into massive necrotic patches, typically surrounded by a yellow halo, resulting in premature leaf fall. The infection also damages the stem, which exhibits dark-striped cracking. Dark green, water-soaked regions are the major signs of a pod infection since that’s where the fungus enters the seeds, coloring and shriveling them. BLB is transmitted through seeds, wind-driven rain, and insects (Zandjanakou-Tachin et al. 2007). Weeds and crop debris from infected areas may potentially serve as sources of inoculum (Sikirou and Wydra 2004).

The cowpea mosaic virus (CpMV) is another serious disease and causes yield loss up to 80–100% (Chant 1959; Gilmer et al. 1974; Williams 1977). CpMV with a single-stranded RNA particle belongs to the genus Como virus in the family Comoviridae (Fenner 1976). The virus is transmitted through seed, sap inoculation, and aphids (Aphis fabae, A. gossypii, and Macrosiphum onobrychis), causing conspicuous mosaic patterns, leaf distortion and deformation, decreased leaf area, flower production, and stunted growth (Van Hoof 1963; Yu 1946).

Among different disease management strategies, utilizing genetic resistance through creating and deploying disease-resistant cultivars is a cost-effective, eco-friendly, and sustainable disease management strategy for cowpea farmers who lack access to resources (Emechebe and Shoyinka 1985; Ahn 1994; Huynh et al. 2018). Research advancements in genomics have made it possible to develop and deploy molecular markers in crop improvement (Varshney et al. 2018; Bi et al. 2018; Mannur et al. 2019). Marker-assisted backcross breeding (MABB) and marker-assisted gene pyramiding schemes (MAGP), which involve the use of molecular markers in conjunction with linkage maps to improve a target trait, are the most employed marker-assisted selection (MAS) strategies in plant breeding.

Marker-assisted backcross breeding (MABB), a simple form of marker-assisted selection, is used extensively in molecular breeding. Transfer of the target gene from the donor parent with accelerated recovery of the recurrent parent genome (RPG) and recurrent parent phenome (RPP) is achieved by molecular and phenotypic selection means. The MABB strategy works by transferring a specific allele at the target locus from a donor line to a recipient line while selecting against donor introgressions across the rest of the genome (Vikas et al. 2013; Boukar et al. 2020; Dormatey et al. 2020). Marker-assisted gene pyramiding depends upon the number of contributing loci, the number of parents, heritability of target genes/QTLs, marker-trait associations, and the duration needed for the transfer of genes. Successful introgression of multiple QTLs/genes and their effects on agronomically superior genotypes have been demonstrated in numerous crop species, including wheat, barley, rice, soybean, chickpea, common bean, and many other crop plants (Hittalmani et al. 2000; Richardson et al. 2006; Jiang et al. 2007; Wang et al. 2012; Zhu et al. 2012; Varshney et al. 2013, 2018; Lucas et al. 2015; Luo et al. 2016; Dinesh et al. 2016, 2018; Varshney 2016; Parhe et al. 2017; Kiryowa et al. 2021).

Genomic resources in the form of genetic maps (Fatokun et al. 1993, 1997; Menendez et al. 1997; Ubi et al. 2000; Kelly et al. 2003; Omo-Ikerodah et al. 2008; Wellington et al. 2009; Agbicodo et al. 2010; Andargie et al. 2011; Xu et al. 2011; Lucas et al. 2015; Gioi and Boora 2012; Dinesh et al. 2016, 2018) and genes/QTLs for resistance to BLB (Dinesh et al. 2016; Kelly et al. 2003; Agbicodo et al. 2010), cowpea mosaic virus (Dinesh et al. 2018; Ouédraogo et al. 2002), cowpea yellow mosaic virus (Gioi and Boora 2012), and cowpea golden mosaic virus (Rodrigues and Santos 2012) are now available for cowpea improvement. In cowpea, a genomic region known as blb-1 assigns the resistance to bacterial leaf blight (BLB) (Dinesh et al. 2016), and the gene linked to cowpea mosaic virus (CpMV) resistance was identified (Dinesh et al. 2018). The variety C-152 is very popular in south India for its wide adaptability and high yield. However, it has become susceptible to major diseases like BLB and CpMV. Therefore, the pyramiding/introgression of blb-1 and CpMV resistance genes into high-yielding adapted cowpea cultivar C-152 through MABB was performed in the present study, and we report the development of an improved version of C-152 with enhanced yield and resistance to BLB and CpMV.

Materials and methods

Genetic material

The cultivar C-152 is a high-yielding mega variety developed from the Iranian cultivar P42 by the University of Agricultural Sciences, Bengaluru. It was released for cultivation in the southern (peninsular) zone of India. The traits, viz. high-yielding potential, semi-spreading habitat, medium brown type seeds, and 90–100 maturity days, made C-152 highly suitable for mixed and cover cropping in plantation crops. However, over a period, C-152 has become susceptible to the CpMV and BLB diseases. Therefore, to improve the resilience of the variety for diseases, it was used as a recurrent parent, and V-16 (resistant to BLB) and V-57817 (resistant to cowpea mosaic virus disease) genotypes were used as donor parents for incorporation of QTL conferring resistance to BLB (blb-1) and CpMV resistance genes, respectively. V-16 is a released variety from ICAR-Indian Agricultural Research Institute (IARI), New Delhi, tolerant to bacterial leaf blight and fungal diseases, red seeded, and adapted to all agro-ecological zones in India (Dinesh et al. 2016). While V-57817, a breeding line developed by IARI, New Delhi, is resistant to the cowpea mosaic virus (Dinesh et al. 2018).

Marker assisted simultaneous and Stepwise backcross breeding (MASSBB)

The major QTL blb-1 for bacterial blight resistance, which explains 30.58% phenotypic variation (Dinesh et al. 2016) and the cowpea mosaic resistance gene (Dinesh et al. 2018) was introgressed into the recipient parent (C-152) through the MABB scheme. Two independent crosses, viz., C-152 × V-16 and C-152 × V-57817, were made to transfer the QTL blb-1 and resistant gene to CpMV, respectively, into C-152. The true F1 plants were identified using a hybridity test based on blb-1 linked CISP markers VuMt401 and VuMt397 and SSR markers MA15 and MA80 linked to CpMV resistance, respectively. The heterozygous plants possessing desirable QTL/allele of the gene from each of the crossing series were independently backcrossed with the recurrent parent to produce BC1F1 and BC2F1 plants and advanced the lines to BC2F4 generation in each cross. Two independent BC2F4 lines from each cross possessing blb-1 and cowpea mosaic resistance gene with maximum genome recovery of the C-152 were identified with the aid of linked markers. These lines were intercrossed to generate intercross F1s (ICF1) to combine the blb-1 and cowpea mosaic resistant gene into the background of C-152 (blb-1 + cowpea mosaic resistance gene). From the 18 positive ICF1 plants for blb-1 and cowpea mosaic resistance, 12 ICF1 plants were selfed to produce 383 F2 plants. The twelve homozygous plants for both blb-1 and cowpea mosaic resistance genes were identified in F2 generation and advanced up to F4 generation through pedigree selection. Background analysis of the advanced superior lines was carried out to quantify the recurrent parent genome (RPG) recovery. These elite plants were then evaluated for their reaction against BLB and CpMV diseases under artificial inoculation as well as at two locations (V. C. Farm, ZARS, Mandya, and GKVK, University of Agricultural Sciences Bangalore) with heavy disease incidence. The schematic representation of the MASSBB is given in Fig. 1.

Fig. 1.

Fig. 1

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Scheme showing marker assisted simultaneous and stepwise backcross breeding (MASSBB) for introgression of blb-1 and cowpea mosaic disease resistance genes

Isolation of genomic DNA, PCR amplification and marker genotyping

Genomic DNA was isolated from young leaves collected from 20–25-day old seedlings by following the CTAB method (Sambrook et al. 2001). The quality and quantity of the DNA sample were checked using 0.8% agarose gel. PCR amplification of markers used for foreground selection and background selection was performed as mentioned in www.cowpeagenomics.med.virginia.edu/CGKB. The PCR mixture contained 20-25ng template DNA, 5 pmol of each primer, 1.0 mM dNTPs, 10x PCR buffer (10 mM Tris, pH 8.4, 50 mM KCl, 1.8 mM MgCl2), and 0.3 u/µl of Taq DNA polymerase (Bangalore Genei Pvt. Ltd., Bengaluru, India) in a reaction volume of 15 µl. Template DNA was initially denatured at 94 °C for 5 min, followed by 35 cycles of PCR amplification with the following parameters: 1 min, denaturation at 94 °C, annealing at 56 °C for 30 s, and primer extension at 72 °C for 2 min, followed by final extension of 72 °C for 10 min. Amplified products were resolved on 3.5% agarose gel containing Ethidium bromide (0.5 µg/ml) using 1X TBE (Sambrook et al. 2001). Gel was allowed to be cast before removing the comb. Loading dye of 5 µl was added to 15 µl of PCR products and mixed well before loading into the wells. Electrophoresis was carried out at a constant voltage of 120 V for 2.5 h. Gels were visualized under UV light, and the DNA pattern was documented using AlfaDigiDoc RT2 unit.

Molecular marker analysis

Marker-assisted foreground selection

The CISP markers (VuMt401 and VuMt397) linked to QTL blb-1 (Dinesh et al. 2016) and another two SSR markers (MA15 and MA80) linked to cowpea mosaic resistance gene (Dinesh et al. 2018) were used for foreground selection in different backcross and advanced generations (Table S1).

Marker-assisted background selection

Based on earlier studies, a total of 72 markers (40 CISPs and 32 SSRs) (Dinesh et al. 2018) uniformly spanning the entire cowpea genome (11 linkage groups) were used to identify plants with maximum recovery of the C-152 parent genome (Table S2).

Disease screening of improved lines under artificial inoculation

Selected lines, homozygous for the CpMV resistance gene and blb-1, along with their parental lines, were grown in earthen pots. Seedlings were separately inoculated with bacterial culture for induction of BLB and sap containing mosaic virus particles for CpMV disease development (Dinesh et al. 2016) under artificial conditions at V.C. Farm, Mandya, Karnataka, India. The bacterial isolates were collected from the infected leaves in the field, and the colonies were isolated and maintained as pure culture under laboratory conditions. Colonies were multiplied, separated, and concentration adjusted to an optical density (OD) of 0.06 corresponding to 108 CFU/mL. Plants were inoculated with bacterial suspension by injecting at the abaxial surface of the first three trifoliates until water-soaked spots appeared using a needleless syringe (Okechukwu 2004). After inoculation, plants were maintained under the greenhouse condition, where the relative humidity was maintained above 80% to ensure disease development. Disease severity was visually scored 20 days after inoculation on the infected leaves (Agbicodo et al. 2010; Wheeler 1969). Cowpea mosaic virus sap, extracted from the infected young leaves of 15 to 20 days old cowpea plants showing characteristic mosaic symptoms maintained in an ice-cold condition, was used as a standard inoculum. The prepared fresh sap was used on the same day for inoculation. The inoculum was applied gently on the upper surface of the leaves along with celite (600 mesh at the rate of 0.025 g/ml) as an abrasive to create sub-lethal wounds with a small piece of absorbent cotton wool. The inoculated leaves were washed 1–2 min after inoculation to remove the excess inoculum with a fine jet of distilled water from a pressurized sprayer, and plants were kept under observation for 15–20 days in the greenhouse. The plants were grouped into different categories as resistant or susceptible, as suggested by Diwakar and Mali (1976).

Field screening of MASSBB introgression lines

The ICF4 progenies, along with recipient and donor parents, were evaluated in a randomized complete block design with three replications. Each progeny was planted in two rows of 5 m length with a spacing of 60 cm between rows and 15 cm between plants. Progenies were artificially inoculated with bacterial suspension as described earlier (Okechukwu et al. 2000) for BLB and by sap inoculation for mosaic disease (Dinesh et al. 2018) development. The BLB disease severity was visually scored 20 days after inoculation (DAI) on the infected leaves using a severity scale of Agbicodo et al. (2010). Further, the percent disease index (PDI) was calculated (Wheeler 1969). The Cowpea mosaic virus produced mosaic symptoms in 8–12 days after inoculation. The disease severity of each progeny was assessed regularly at 15-day intervals as percent disease incidence on 10 plants. The disease scoring was made as per Diwakar and Mali (1976).

Evaluation of MC 17−2 (KBC-12) in the trials conducted by ICAR-AINP on arid legumes in South India

The line MC 17−2 (KBC-12) with 97.90% genomic recovery from C-152 with high grain yield in the station trials was proposed for evaluation in the initial varietal trial (IVT) in South India by ICAR-AINP on Arid Legumes during the rainy season of 2018. Each entry was planted in four rows of 4 m length with a spacing of 45 cm between rows and 15 cm between plants. It was promoted for evaluation in AVT-1 (during the rainy season of 2019) and AVT-2 (during the rainy season of 2020) based on its yield superiority in IVT.

Phenotypic evaluation of MC 17−2 (KBC-12) in multilocation trials

Eight genotypes that consisted of MC 17−2 (KBC-12) and other promising entries were evaluated in multilocation trials at Zonal Agricultural Research Station (ZARS), GKVK, Bangalore; Agricultural Research Station (ARS), Chintamani; ARS, Balajigapade; and ARS, Pavagada in Zone-5, ZARS, Mandya; and ARS, Kunigal in Zone 6, employing a randomized complete block design with three replications during the rainy season of 2022. Each genotype was planted in four rows of 4 m length with a spacing of 45 cm between rows and 10 cm between plants.

Assessment of stability of KBC-12 in multi-location trials

The phenotypic stability of KBC-12, along with seven other potential cowpea lines, was assessed across six locations covering zones 5 and 6 of Karnataka. A pooled analysis across environments was carried out to deduce the variance components of different sources of variation and to detect the presence of GEIs. The AMMI ANOVA was performed to partition GEIs to different PCs (Gauch and Zobel 1988; Nataraj et al. 2021) using “metan” package available in R version 4.4.3. Genotype + Genotype × Environment (GGE) biplot analysis consisting of (1) which won where that enables to recommend genotypes to specific location and (2) mean vs. stability to assess the stability of the genotype were also performed.

Evaluation of MC 17−2 (KBC-12) in farm trials in Southern Karnataka

The genotype KBC-12 was evaluated in farm trials in Zone 5 and 6 of Karnataka, along with the best check variety KBC-9 on farmers’ fields. Each entry was planted in a 500 m2 area with 45 cm between rows and 15 cm between plants during the 2023-24 rainy season.

Results

Marker-assisted simultaneous and Stepwise transfer through backcross breeding (MASSBB)

Marker-assisted backcross breeding was adopted to incorporate blb-1 QTL conferring resistance to BLB and the cowpea mosaic resistance gene into an elite cultivar C-152 through two separate backcrossing schemes (Fig. 1).

Marker assisted introgression of BLB resistance QTL (blb-1)

For transferring blb-1 QTL, F1 plants from the cross C-152 × V-16 were confirmed for hybridity using QTL blb-1 linked markers (VuMt401 and VuMt397), and 19 F1 plants were then backcrossed to C-152 to produce 63 BC1F1 seeds. These sixty-three BC1F1 plants were subjected to foreground selection using markers (VuMt401 and VuMt397) flanking the blb-1, and 17 plants were found to be heterozygous for the blb-1. Plants with maximum RPG recovery and heterozygous alleles for blb-1 QTL were then backcrossed to C-152 to produce 51 BC2F1 plants. Selected plants from 51 BC2F1 based on foreground and background analysis were advanced to produce BC2F2 seeds. Foreground and background selection were continued till the BC2F4 generation. Plants found homozygous for blb-1 with maximum RPG recovery at BC2F4 were selected for developing inter cross F1 (ICF1).

Marker assisted introgression of CpMV resistance

Concurrently, F1 plants from another cross, C-152 × V-57817, were generated, and hybridity was validated for 17 plants using molecular markers (MA15 and MA80) associated with the cowpea mosaic resistance gene. F1 plants with the cowpea mosaic resistance gene were backcrossed to C-152 to generate 33 BC1F1 plants where foreground selection was practiced. A total of 23 plants were identified to be heterozygous for marker linked to mosaic resistance. These plants were then crossed with the recurrent parent C-152 to produce BC2F1 seeds, which were then subjected to foreground selection. Further, 69 plants found heterozygous for the mosaic resistance gene with maximum RPP recovery were backcrossed to produce BC2F2 seeds and subjected to foreground and background analysis. The 16 BC2F3 plants generated were subjected to phenotypic selection and advanced to BC2F4. Foreground and background selections were continued till BC2F4 generations. Plants found homozygous for the cowpea mosaic resistance gene with maximum RPG recovery at BC2F4 were selected for developing inter cross F1 (ICF1).

Development of intercrossed filial generations for BLB and CpMV resistance

Superior BC2F4 plants from both backcrossing schemes that were homozygous for the cowpea mosaic resistance gene and blb-1 were intercrossed and produced 18 intercross (ICF1) plants. Heterozygous plants for the blb-1 and cowpea mosaic resistance gene were tagged using foreground analysis with the corresponding gene and QTL-linked markers. Out of 18 ICF1, 12 plants were forwarded to produce a sizable F2 population with 383 plants. Based on phenotypic analysis, 236 F2 plants were selected and subjected to foreground analysis with VuMt401, VuMt397, MA15, and MA80 markers, which identified 26 plants homozygous for both the blb-1 and the CpMV resistance gene. These plants were advanced using the pedigree method and identified 12 best performing intercross ICF3 families that were homozygous for blb-1 and cowpea mosaic resistance gene (Fig. 1) with maximum recovery of RPG.

The background analysis of 12 selected ICF3 lines was performed using a set of 72 markers (40 CISPs for BLB and 32 SSRs for CpMV), four to six markers per linkage group that were evenly distributed across all 11 cowpea chromosomes (Table S2). The percent of recurrent parent genome recovery ranged from 85.41 to 97.90%. The average recurrent parent genome recovery percentage of 92.91% was recorded. The 10 selected lines, along with C-152 and another check variety for yield (KBC-2), were evaluated during the rainy season of 2016 at ZARS, V. C. Farm, Mandya, for their response to BLB and CpMV and yield (Table 1). These 10 superior lines having high resistance were designated as MC 17−1, MC 17−2, MC 17−3, MC 17−4, MC 17−5, MC 17−6, MC 17−7, MC 17−8, MC 17−9, and MC 17−10.

Table 1.

Mean performance of cowpea lines for grain yield and reaction to cowpea mosaic virus and bacterial leaf blight at V.C. Farm, Mandya in Karnataka during 2016

Genotypes Grain yield (kg/ha) Yield advantage (%) Disease reaction % RPG
Over check KBC-2 (%) Over recurrent parent C-152 (%) CpMV BLB
MC-17-1 1236 21.70 39.70 0 I 2.2 R 97.90
MC 17−2 (KBC 12) 1362 34.20 53.90 0 I 0 R 97.90
MC-17-3 1241 22.30 40.20 4 R 6.4 R 91.67
MC-17-4 1259 24.00 42.20 0 I 0.6 R 95.83
MC-17-5 951 −6.30 7.40 12 MR 28.4 TR 87.50
MC-17-6 1246 22.70 40.80 0 I 3.6 R 95.83
MC-17-7 802 −21.00 −9.40 18 MS 60.2 S 87.50
MC-17-8 1289 27.00 45.60 0 I 1.4 R 97.90
MC-17-9 978 −3.60 10.50 22 MS 58.2 S 85.41
MC-17-10 998 −1.70 12.80 14 MR 27.6 TR 91.67
KBC-2 (Ch) 1015 - - 68 S 42 S -
C-152 (RP) 885 - - 62 S 34 S -
CV (%) 9.29 - - - - - - -
LSD (0.05) 236.27 - - - - - - -
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Ch check variety, RP recurrent parent, CV coefficient of variation, LSD least significant difference, CpMV cowpea mosaic virus, BLB bacterial leaf blight, RPG recurrent parent genome

I Immune, R Resistant, MR Moderately Resistant, MS Moderately Susceptible, S Susceptible

Phenotypic evaluation of introgression lines in station trial

BLB and CpMV screening revealed that 26 of 383 ICF2 plants were resistant to BLB and CpMV. These 26 resistant lines were advanced to produce ICF3 families, which were then subjected to background analysis and identified 12 progenies with the highest RPG recovery (> 80%) which were then forwarded to generate ICF4 lines. Screening for BLB and CpMV revealed that 10 of 12 ICF4 lines were consistently resistant to BLB and CpMV (Table S3). Based on the response of 10 genotypes out of 12, to diseases and grain yield in station trial conducted during the rainy season of 2016, the six top performers, viz., MC 17−1, MC 17−2, MC 17−3, MC 17−4, MC 17−6, and MC 17−8, were identified for further multi-environment evaluation (Table 2). All six selected lines were phenotyped for grain yield as well as disease reaction for BLB and CpMV during the rainy season of 2017 in two environments, viz. Mandya and Bengaluru (Table 2). In the multi-location trial, MC 17−2 (KBC-12), MC 17−4, MC 17−1, and MC 17−8 were the best performers with a mean yield advantage of 18, 15, 14, and 11%, respectively over the best check variety KBC-2 and mean grain yield superiority of 17, 15, 13 and 10% respectively, over the recurrent parent C-152. The percent disease incidence index (PDI) for CpMV for these four genotypes was zero over both locations. Furthermore, the PDI for BLB was 0, 0.8, 1.4, and 2.2%, respectively, for MC 17−2, MC17-4, MC 17−8 and MC 17−1. Nevertheless, MC 17−3, and MC 17−6 exhibited varied responses to both diseases. The MC 17−3 manifested a moderate susceptibility and tolerant reaction to CpMV at Bengaluru and Mandya locations. Similarly, MC 17−6 displayed an immune response to CpMV at Mandya, whereas it showed moderate resistance at Bengaluru. Furthermore, this genotype documented a resistant reaction at Mandya, and tolerant reaction at Bengaluru for BLB. Due to this unreliable and non-stable disease response, we eliminated MC 17−3 and MC 17−6 genotypes from further testing.

Table 2.

Performance of cowpea lines for grain yield and disease reaction at Mandya and Bengaluru in Karnataka during rainy season of 2017

Genotypes Grain yield (kg/ha) Yield advantage (%) Disease reaction at Mandya Disease reaction at Bengaluru % RPG
CpMV BLB CpMV BLB
Mandya Bengaluru Mean Over check KBC-2 Over recurrent parent C-152 PDI (%) Disease response PDI (%) Disease response PDI (%) Disease response PDI (%) Disease response
MC-17-1 953 1467 1210 14 13 0 I 2.2 R 0 I 2.4 R 97.90
MC-17-2 (KBC-12) 995 1514 1255 18 17 0 I 0 R 0 I 0 R 97.90
MC-17-3 720 953 837 −21 −22 16 MS 24 TR 20 MS 20 TR 91.67
MC-17-4 967 1494 1231 15 15 0 I 0.8 R 0 I 0.6 R 95.83
MC-17-6 908 1020 964 −10 −10 0 I 3.6 R 12 MR 28 TR 95.83
MC-17-8 938 1419 1178 11 10 0 I 1.4 R 0 I 1.6 R 97.90
Checks
KBC-2 (Ch) 716 1415 1066 - - 68 S 32 S 56 S 38 S
C-152 (RP) 870 1279 1075 - - - - - - - - - -
CV (%) 9.02 8.35 - - - - - - - - - - -
LSD (0.05) 193.72 267.98 - - - - - - - - - - -
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Ch check variety, RP recurrent parent, CV coefficient of variation, LSD least significant difference, CpMV cowpea mosaic virus, BLB bacterial leaf blight, RPG recurrent parent genome, PDI per cent disease index, I immune, S susceptible, MS moderate susceptible, R resistant, MR moderate resistant, TR tolerant reaction

Evaluation of MC 17−2 (KBC-12) in the trials conducted by ICAR-AINP on arid legumes in South India

In the Initial Varietal Trial (IVT) of the ICAR-All India Network Project on Arid Legumes during Rainy Season 2018 conducted at eight diverse locations, viz., Bangalore, Pattambi, Coimbatore, Vamban, Dharwad, Madhurai, Warangal, and Pandharpur, the variety KBC-12 (MC 17−2) recorded higher grain yield over the best check in three of the eight environments (Fig. 2a). The overall yield advantage was 8.68, 18.65, and 28.68% over the check varieties DC 15, TPTC 29 and PCP 0306, respectively (Fig. 2b). Similarly, there was a 47.0, 46.90, and 9.30% overall percentage increase in MC 17−2 in the first-year advanced varietal trial (AVT-I) compared to the check varieties TPTC 29, KBC 9 and DC 15, respectively, during 2019-20 (Table 3). Furthermore, MC 17−2 displayed 8.10 and 1.10% yield superiority over the checks TPTC 29 and DC 15 in the second-year advanced varietal trial (AVT-II) during 2020-21 and produced higher/comparable grain yields over the best check variety in eight of the ten environments (Table 4).

Fig. 2.

Fig. 2

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Initial varietal trial of the ICAR-All India network project on Arid legumes conducted during 2018–19 to compare the yield (kg ha–1) (a) performance of the introgression line KBC 12 (MC 17−2) produced in the genetic background of recurrent genotype C-152 at eight locations and (b) average yield across eight locations in Southern India. (Source: ICAR-All India Network Project on Arid Legumes Annual Report 2018-19)

Table 3.

Advanced varietal trial I (first year) of the ICAR-All India network project on arid legumes conducted during 2019-20 to compare the yield (kgha–1) performance of the introgression line KBC 12 (MC 17−2) produced in the genetic background of C-152 with the recurrent genotype at eight locations in Southern India. (Source: ICAR-All India network project on arid legumes annual report 2019-20)

Entries Ananthapuram Bangalore Pattambi Coimbatore Vamban Madhurai Warangal Pandharpur Mean Yield advantage over checks (%) Freq*
MC-17-2 (KBC-12) 1750 2685 1192 1690 934 892 985 1069 1400 - 6/8
KBC 9 (Ch) 1229 1331 1114 1023 430 885 425 1189 953 46.9 -
DC 15 (Ch) 954 2152 1289 1176 1526 779 1481 894 1281 9.3 -
TPTC 29 543 1365 724 1555 653 833 776 1169 952 47.0 -
LSD @ 0.05 266.89 359.86 185.55 172.01 156.75 104.58 213.74 95.18 - - -
CV (%) 19.84 15.36 12.80 9.35 14.22 8.65 18.68 5.12 - - -
Experimental mean (kg/ha) 806 1401 867 1101 772 725 681 1110 - -
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*Frequency indicates the ratio of a number of locations in which the introgression line performs better or comparable than the best check variety to the total number of locations evaluated; Ch check variety, CV coefficient of variation, LSD least significant difference

Table 4.

Advanced varietal trial II (second year) of the ICAR-All India network project on arid legumes conducted during 2020-21 to compare the yield (kgha–1) performance of the introgression line KBC 12 (MC 17−2) produced in the genetic background of C-152 with the recurrent genotype at ten locations in Southern India. (Source: ICAR-All India network project on arid legumes annual report 2020-21)

Entries Bangalore Pattambi Coimbatore Vamban Tirupati Dharwad Madhurai Ananthapuram Warangal Pandharpur Mean Yield advantage over checks (%) Freq*
MC-17-2 (KBC-12) 1282 1721 1465 1332 829 1021 904 551 1125 857 1109 - 8/10
KBC 9 (Ch) 1134 890 1760 1568 1110 1056 744 709 1200 935 1111 - -
DC 15 (Ch) 1148 1011 1519 1660 785 917 796 817 1206 1109 1097 1.10 -
TPTC 29 1167 282 1583 1318 589 1167 781 476 1663 1235 1026 8.10 -
LSD @ 0.05 82.03 124.73 239.69 301.08 300.70 102.40 108.81 140.88 278.59 116.01 - - -
CV (%) 6.33 8.16 11.65 16.56 21.63 5.88 8.24 15.02 13.76 6.45 - - -
Experimental mean (kg/ha) 775 914 1230 1087 831 1042 789 561 1211 1076 - -
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*Frequency indicates the ratio of a number of locations in which the introgression line performs better or comparable than the best check variety to the total number of locations evaluated; Ch check variety, CV coefficient of variation, LSD least significant difference

Phenotypic evaluation of MC 17−2 (KBC-12) in multilocation trials

Multilocation testing of the newly improved cowpea variety KBC-12, along with the check KBC-9, was performed during the rainy season of 2022 in Zones 5 and 6 of southern Karnataka. The genotype MC 17 − 2 (KBC-12) recorded a higher grain yield of 1300 Kg/ha as compared to the best check variety, KBC-9 (1034 kg/ha) in Zone 5 (Fig. 3a). In Zone 6, the test entry KBC-12 exhibited significantly higher grain yield of 2142 kg/ha over the check variety, KBC-9 with 1710 Kg/ha (Fig. 3b). Overall, KBC-12 showed a 19.31% higher grain yield over the check KBC-9.

Fig. 3.

Fig. 3

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Performance of KBC-12 in multilocation testing (a. Zone 5 and b. Zone 6) in Southern Karnataka during rainy season of 2022-23 (Kg/ha). CD: critical difference @ 5% significance and CV: Coefficient of Variation

Stability analysis of KBC-12

Results from the pooled analysis of variance revealed that the mean sum of squares due to location, genotype and genotype by environment were significant (Table S4) indicating the existence of genetic differences among the tested genotypes and influence of environment on the trait expression. Further, AMMI ANOVA revealed that the first two IPCs were significant at p < 0.05 while the rest were nonsignificant. The proportion of variation explained by first and second IPCs were 54.90 and 23.0%, respectively (Table S5). Together, these two IPCs contributed 77.90% of variation implying AMMI model captured significant interaction effects. AMMI1 (Fig. 4) and AMMI2 (Fig. 5) biplots revealed the high yielding and stable performance of KBC-12 across the tested environments.

Fig. 4.

Fig. 4

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AMMI1 biplot for grain yield (q/ha)

Fig. 5.

Fig. 5

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AMMI2 biplot for grain yield (q/ha)

GGE bi-plots derived based on the first two IPCs are used to interpret patterns of genotype × environment interaction, and in identification of the genotypes that are specifically suited to a particular location and those that are stable across locations. Which won where biplot gives a clear idea about which genotype has won in which mega environment. Out of six studied environments, the locations E1 (ARS, Kunigal), E2 (ARS, Balajigapade), E3 (Pavagada), E4 (ARS, Chintamani) and E6 (VC Farm, Mandya) constituted one mega environment while the environment E5 (ZARS, GKVK) alone formed a separate mega environment (Fig. 6). The genotypes KBC-12 and KBC-11 were found to be the winning genotypes in first and second mega environments, respectively. In addition to this, “mean vs. stability” view of the GGE biplot evaluates stability of the genotypes and identified KBC-12 as highly stable (Fig. 7).

Fig. 6.

Fig. 6

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Which-won-where view for grain yield (q/ha)

Fig. 7.

Fig. 7

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Mean vs. stability for grain yield (q/ha)

Evaluation of MC 17−2 (KBC-12) in farm trials in Southern Karnataka

Further, the performance of cowpea test entry KBC-12 in comparison with the best check, KBC-9, was assessed in 40 farm trials across zones 5 and 6 of Karnataka state during the rainy season of 2023-24. The percent increase in yield advantage over the check KBC-9 is given in Table 5. An average of 11.88% and 17.37% improved yield advantage of KBC-12 over KBC-9 was recorded in zones 5 and 6, respectively. KBC-12 at different phenological stages is given in Fig. 8. Disease response of KBC-12 in comparison with the recurrent parent C-152 is given in Fig. 9.

Table 5.

Performance of Cowpea variety KBC-12 in farm trials in zone 5 and zone 6 (kg/ha) of Karnataka during rainy season of 2023-24

KSDA/KVK/EEU KBC-12 KBC-9 (Check) % Increase
(Kg/ha) (Kg/ha)
Zone 6 Total = 17
KVK, Mandya (3) 1247.66 1118.50 11.54
KVK, Chamarajanagar (3) 1094.00 1069.66 2.27
KSDA, Mandya (2) 194.00 174.00 11.49
KSDA, Mysore (3) 750.00 680.00 10.29
KSDA, Chamarajanagar (3) 1218.00 986.66 23.44
EEU, Mysore (3) 1283.33 1143.33 12.24
Mean 964.50 862.03 11.88
Zone 5 Total = 23
KVK, Chinthamani (5) 1535.90 1388.50 10.61
KVK, Ramanagara (4) 1084.00 956.75 13.30
KVK, Doddaballapura (5) 1104.00 988.00 11.74
KSDA, Bangalore rural (3) 1456.66 1073.33 35.71
KSDA, Kolar (2) 1315.00 1107.50 18.73
KSDA, Chikkaballapur (4) 1120.00 974.20 15.00
Mean 1269.26 1081.38 17.37
Overall Mean 1116.88 971.70 14.94
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KSDA Karnataka State Department of Agriculture, KVK Agriculture Science Center, EEU Extension Education Unit; Values in parenthesis indicate number of farm trials conducted

Fig. 8.

Fig. 8

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KBC-12 at different phenological stages

Fig. 9.

Fig. 9

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Disease response of KBC-12 and C-152 (recurrent parent) for a. Bacterial leaf blight and b. Cowpea mosaic virus disease

Discussion

The majority of high-yielding consumer acceptable varieties in various crops are being rejected by the farmers for cultivation due to their susceptibility to biotic and abiotic stresses. This potentially limits the percent adaptability and acceptance of research in the field. C-152, a high-yielding cowpea variety developed by UAS Bangalore was released for cultivation in the southern (Peninsular) zone of India. It developed susceptibility to two major diseases, bacterial leaf blight and cowpea mosaic virus, over a period of prolonged cultivation. Bacterial leaf blight (BLB) and cowpea mosaic virus (CpMV) diseases are the two major diseases of cowpea in tropical, sub-tropical, and semi-arid regions, resulting in significant yield losses (Hammed et al. 2019; Saha et al. 2022; Guillermo et al. 2023). In recent times, these diseases have become major threats due to changing climatic conditions. The chemical control of these diseases is non-economical and ineffective to resource-poor cowpea farmers. Therefore, the exploitation of genetic resistance in cowpea through contemporary breeding approaches provides a farmer with an environment-friendly solution for disease management.

The application of molecular markers in conjunction with phenotype-based selection is known to enhance the selection accuracy, in turn accelerating the rate of genetic gain. Marker-assisted backcrossing, which employs gene-specific and genome-wide markers for foreground and background selection of target gene and recurrent parent genomes, respectively, was employed in this study. It is an environment-independent, accurate, and quick technique for developing disease-resistant genotypes (Bharadwaj et al. 2021).

To pyramid recessive gene for resistance to cowpea mosaic and QTL governing resistance to BLB into a single cultivar through classical breeding approach, the breeding lines are required to be tested for resistance at each backcross generation, which is a daunting and resource-demanding task (Vikas et al. 2013). The MABB in our study identified resistant plants with the help of blb-1 (VuMt401 and VuMt397) and CpMV resistance gene linked (MA15 and MA80) markers without repeated testing during the development of pyramided lines. MABB has previously been successfully employed for improving agronomic traits including disease resistance as well as economically significant traits in cowpea (Lucas et al. 2015), wheat (Jiang et al. 2007), barley (Richardson et al. 2006), rice (Hittalmani et al. 2000; Luo et al. 2016), soybean (Wang et al. 2012; Parhe et al. 2017), chickpea (Varshney et al. 2013, 2018; Varshney 2016), common bean (Kiryowa et al. 2021), cowpea (Lucas et al. 2015; Dinesh et al. 2016, 2018), potato (Zhu et al. 2012) and many other crop plants. Marker-assisted background analysis was performed employing 72 (40 CISPs and 32 SSRs) markers among the superior lines that aided in selection of the MASSBB lines with maximum recovery of RPG as high as 97.90% in MC 12 − 1, MC 12 − 7, and MC 12 − 8; 95.83% in MC 12 − 3, and MC 12 − 5 in early generation (ICF4) in the genetic background of the C-152 variety. This remarkable recovery of RPG was possible due to the contribution of recurrent parent alleles from both the backcross progenies (C-152 + CpMV resistant gene and C-152 + blb-1), which resulted in the fixation of recurrent parent alleles in inter crossed cowpea lines. It is evident from the results that phenotypic selection for disease resistance (BLB and CpMV) and yielding potential of C-152 coupled with marker-assisted background analysis led to rapid recovery of the C-152 genome and phenome. The improved lines with the CpMV resistance gene and blb-1 QTL in homozygous condition exhibited a similar kind of resistance to CpMV and BLB diseases as that of the resistant donors, V-57187 and V-16. Similarly, about 97% of the recurrent parent (Moussa Local) genome was recovered as early as in BC2 generation by employing the MABC method with SNP markers in an attempt to introgress QTLs for drought tolerance, striga resistance, and root-knot nematode resistance in cowpea (Batieno et al. 2016). The use of MASSBB enabled the recovery of 95% RPG recovery in ICF2 and ICF3 generations, which could otherwise be achieved only in ICF6 and ICF7 through a classical breeding approach. Attamah et al. (2024) reported the RPG recovery ranging from 72% (ICF2_Zaa/556/SARC-P47) to 93.80% (ICF2_Zaa/556/SARC-P6) with an average RPG recovery of 87% while pyramiding aphid resistance in the elite cowpea variety Zaayura in Ghana. Batieno et al. (2016) opined that the recovery of the recurrent parent background with minimum cycles of backcrossing allowed early selection and helped to minimize population sizes at each generation, thereby cutting down the resource and time needed in varietal improvement. Similar proportion of recurrent parent genome recovery was earlier reported by Varshney et al. 2014a, b; Mannur et al. (2019); Bharadwaj et al. (2021), (2022) in chickpea; Basavaraj et al. (2010) and Vikas et al. (2013) in rice.

Preliminary field evaluation of 10 cowpea lines (MC 17−1, MC 17−2, MC 17−3, MC 17−4, MC 17−5, MC 17−6, MC 17−7, MC 17−8, MC 17−9, and MC 17−10) was conducted at Mandya during the rainy season of 2016 to validate their reactions to BLB and mosaic disease. Six lines (MC-17-1, MC-17-2, MC-17-3, MC-17-4, MC-17-6, and MC-17-8) with the highest level of RPGR (91.67–97.90%) exhibited a high level of resistance (immune and resistant) to both diseases compared to the disease incidence in the susceptible C-152 recurrent parent and susceptible check KBC-2. Out of these six superior performing lines, four lines (MC 17−1, MC 17−4, MC 17−8, and MC 17−2 (KBC-12)) were identified as stable for resistance reaction at two locations (Mandya and Bangalore) during the rainy season of 2017 and exhibited yield advantages of 10–17% over the C-152 (recurrent parent) and 11–18% over the KBC-2 (check variety).

The top-performing and stable line KBC-12 for yield and resistant reactions to diseases at Mandya and Bangalore during 2016 and 2017 was proposed for evaluation in the Initial Varietal Trial (IVT) of the ICAR-All India Network Project on Arid Legumes during 2018-19 to evaluate its performance at the national level across eight locations. The mean yield of KBC-12 (MC 17−2) was superior (1126 kg/ha) over the three checks DC-15 (1036 kg/ha), TPTC-29 (949 kg/ha) and PCP 0306 (875 kg/ha) with a yield advantage of 8.68%, 18.65%, and 28.68%, respectively. Additionally, this outstanding line was evaluated in Advanced Varietal Trials (AVT-I and AVT-II) under the ICAR-All India Network Project on Arid Legumes for two years at eight and 10 locations in 2019-20 and 2020-21, respectively, to determine its ability to perform at the national level for potential release as an improved variety for commercial farming. KBC-12 (MC 17−2) ranked first in AVT-I with the highest mean yield of 1400 Kg/ha which corresponds to a 9.29% mean increase in grain yield over the best check variety, DC-15, with superior performance in AVT-II (1109 Kg/ha) over the three checks, KBC-9, DC-15, and TPTC-29.

The newly developed cowpea line MC 17−2 (KBC-12) was found promising in multilocation trials covering Zones 5 and 6 of Karnataka state. Results from pooled and AMMI analysis, revealed the existence of genotype by environment interactions. Thus, stability of KBC-12 in the six environments falling in Zone 5 and 6 was assessed by employing AMMI and GGE biplot analysis. Results from AMMI (AMMI1 and AMMI2 biplots) and GGE biplot (which won where and mean vs. stability analysis) indicated the higher stability and yielding potential of KBC-12 across six environments (Gauch 2013; Mbeyagala et al. 2021; Aleme et al. 2024).

Further, the promising test entry KBC-12 was evaluated in 40 farm trials across Zones 5 and 6 of Karnataka. It outperformed the check KBC-9 in both zones, indicating its yielding potential with resistance to bacterial leaf blight and cowpea mosaic virus diseases, as it possesses the beneficial alleles from both the donor parent and the background of popular, well-adapted recurrent parent C-152.

Based on its overall performance, KBC-12 has been released for commercial cultivation in southern India during 2024. The seeds of the new variety KBC-12 were deposited in the National Bureau of Plant Genetic Resources, New Delhi, and assigned the National Identity number IC652010.

Conclusion

Most of the MABB studies were confined to the incorporation of resistance genes from a single donor source into the recurrent parents. Here, we report the practical application of MASSBB for introgression of the cowpea mosaic resistant gene and QTL blb-1 from two different donors, V-57187 and V-16, respectively, into the single recurrent parent, C-152 by cautious management of time and resources. A higher proportion of recurrent parent genome was recovered in early generations with target resistance genes from the donor parents. The line KBC-12 (MC 17−2) demonstrated superior grain yield advantage over the best checks, in different trials. This improved line with enhanced resistance has been released as a variety for commercial cultivation in Southern India by the Central Sub-Committee on Crop Standards, Notification and Release of Varieties of Agri-Horticultural Crops and can also be used as a breeding line.

Electronic supplementary material

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Supplementary Material 1 (12.7KB, docx)
Supplementary Material 2 (23KB, docx)
Supplementary Material 3 (16.3KB, docx)
Supplementary Material 4 (14.6KB, docx)
Supplementary Material 5 (15.1KB, docx)

Author contributions

H.C.L. and K.P.V. conceived and designed the study, H.B.D., S.C.T., T.V.K., P.S. and D.S.A., performed the experiments. H.C.L., S.C.T., M.G.M. and M.S.S., wrote the manuscript. All authors discussed the results and commented on the manuscript.

Data availability

All data generated or analyzed during this study are provided in this published article and its supplementary data files.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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