Application of marker-assisted backcrossing to improve cowpea (Vignaunguiculata L. Walp) for drought tolerance

Molecular-assisted backcrossing (MABC) was used to introgress drought tolerance, Striga and rootknot nematode resistance QTLs into a farmer-preferred widely grown cowpea landrace adapted for intercropping in Burkina Faso. Two backcross populations were developed using two drought tolerant donor lines IT93K-503-1 (nematode resistant) and IT97K-499-35 (Striga resistant) and the drought sensitive landrace Moussa Local as the recurrent parent. A set of 184 genomewide EST-derived SNP markers spanning an average of 2-cM intervals and flanking known trait positions was employed for genotyping the backcross progenies using the cowpea KASP genotyping platform. BC1F1 individual plants that were heterozygous for SNPs associated with drought tolerance, Striga and/or nematode resistance (foreground SNPs) and carried as many recurrent-parent alleles as possible at other SNP loci (background SNPs) were selected for the next backcross cycle. This process was repeated to produce BC3F1 families of each donor population. The six best families from the two donors based on marker aided selection and preliminary yield performance under well -watered and water-restricted field trials and Striga resistance screening were selfed to increase seed (BC3F2) for further yield tests. This study demonstrated the high efficiency of using SNP markers in foreground and background marker selection in a MABC scheme to improve a widely grown cowpea variety by adding drought tolerance and biotic stress resistance traits.


INTRODUCTION
Modern plant breeding based on the fundamental principles of inheritance has become an important component of agricultural science and technology.Conventional breeding methodologies have proved to be *Corresponding author.E-mail: bbjersi2003@yahoo.fr.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License broadly successful in development of plant cultivars and germplasm.However, conventional breeding is still dependent to a considerable extent on subjective evaluation and empirical selection using often highly variable and time-consuming phenotyping assays.Recent technological advances in molecular markerassisted breeding (MAB) have brought new opportunities and prospects for optimizing the accuracy and efficiency of breeding systems through indirect selection.
In backcross breeding, the main objective is the introgression of one or more genes from a donor into the background of an elite variety and to recover the recurrent parent genome as rapidly as possible (Semagn et al., 2006).In the past, this was usually achieved by conventional backcross methods, but in many ways the same objective is being pursued through transgenic breeding, bypassing recombination altogether but introducing a value-added trait.Recurrent backcrossing is thus a traditional breeding method commonly employ ed to transfer alleles at one or more loci from a donor to an elite variety (Allard, 1960).This method requires many generations over several years before rec overing the background of the elite cultivar.According to Semagn et al. (2006), the recovery of 99.2% of the elite cultivar could take at least six generations when t here is no deviation.In cases involving a deviation, Y oung and Tanksley (1989) for example found an introgressed segment as large as 4 centiMorgans (cM) in tomato cultivars developed after 20 backcrosses, and one cultivar developed after 11 backcrosses still contained the entire chromos ome arm carrying the gene from the donor parent.
During the past two decades, development of genomic resources has facilitated gene transfer using molecular markers.The use of SNP markers in MAB programs has progressed rapidly together wit h development of technologies and plat forms for the discovery and highthroughput (HTP) screening of S NPs in many crops (Mammadov et al., 2012).The MAB technology allows transfer of target genome regions coupled with extensive genetic mapping and QTL discovery for the development of molecular markers for use in mark er-assisted backcrossing (MAB C) and marker-assisted selection (MAS) (Semagn et al., 2006).Molecular mark ers are tools that can be used as chromosome landmarks to facilitate the introgression of genes associated with economically important traits.It is an approach that has been developed to avoid problems connected with conventional plant breeding by shifting the selection criteria from selection of phenotypes towards selection of genes that control traits of interest, either directly or indirectly.Unlike conventional backcross breeding, MAB can be viewed as a four -step selection process to quickly recover the recurrent parent genotype (Frisch et al., 1999).This includes: (1) selecting individuals carrying the targeted alleles, (2) selecting individuals homozygous for the recurrent parent genotype at loci flanking the target locus, (3) selecting individuals homozygous for recurrent parent genotype at remaining loci on the same chromosome comprising the target ed allele, and (4) selecting individuals that are homozygous for the recurrent parent genotype at most genotyped loci across the whole genome among those that remain.
The use of MAS for introgression of major quantitatively inherited trait loci for stress tolerance is increasingly being applied in crop improvement.However, the use of such technology has been slow in pulse breeding programmes (K umar et al., 2011).Effort are made for t he use of high-throughput genotyping platforms in pulses like chickpea, common beans and cowpea (Muchero et al., 2008;Muchero et al., 2009a;Muchero et al., 2009b).
In this study, the MABC method has been used to introgress drought-related QTLs (Muchero et al., 2009a;Muchero et al., 2009b), striga and nematode resistance genes from two IITA cowpea varieties into one local farmer-preferred variety.

Leaf sam pling and DNA extraction
A sampling kit ( LGC Genomics, Teddington, UK) w as used w hich is designed to facilitate both cutting of leaf discs and their transport and concomitant des iccation, for eventual DNA isolation.For each sample, 4 leaf discs of 6-mm-diameter w ere cut using the Harris Uni-Core leaf-cutting tool supported by the Harris self -healing cutting mat and placed into one w ell of a 96-w ell storage plate, whereupon the plate w as sealed w ith a perforated (gas -permeable) heat seal by apply ing a medium hot household ir on to t he top of the seal for about 2 seconds.The sealed plate w as then placed in a heavy-duty sealed bag in the presence of a desiccant to dehydr ate and pr eserve the leaf tissue dur ing transit at ambient temperature.Decontamination of the leaf -cutting tool betw een sampling different plants w as achieved us ing 70% ethanol or 2% sodium hypochlorite.The leave samples w ere sent to LGC Genomics for DNA extraction and SNP genotyping using the KASP system.

Selection of m arkers
A set of 184 genome w ide SNP markers spanning an average of 2-cM intervals w as selected using the BreedIt © SNP Selector tool (http://br eedit.org/)developed at University of California -Riverside (UCR).The SNP Selector provides an interface to gener ate customized lists of SNPs based on c M distance betw een markers genome-w ide, and markers flanking know n trait positions.All SNP markers w ere developed from EST of drought-stressed tissues, so there is a chance the markers are associated w ith drought tolerance candidate genes (Muchero et al., 2009b).

Plant m aterials and QTL introgression procedures
Tw o drought-tolerant lines from IITA ( IT93K-503-1 and IT97K-499-35) that w ere found to be drought tolerant in Burkina Faso (Saw adogo, 2009) based on their yielding and staying green abilities under w ater stress conditions and in w hich drought-toler ant QTL have been discovered and mapped ( Muchero et al., 2008;Muchero et al., 2009a;Muchero et al., 2009b;Muchero et al., 2010;Muchero et al., 2011) w ere used as donors of positive alleles of drought tolerance QTLs, and Str iga and root-knot nematode resistance genes.'Moussa Local', a local farmer-preferred pur ified variety from Burkina Faso, w as used as the recurrent parent.The donor alleles for yield and nematode resistance w ere selected based on results from UCR/INERA collaborative on-going pr ojects.The donor alleles for Str iga res istance w ere selected based on synteny w ith the Str iga resistance locus reported in Ouedraogo et al. (2002).
For the MABC scheme, IT93K-503-1 and IT97K-499-35 w ere crossed to Moussa Local to obtain F1s.The F1s w ere backcrossed to Moussa Local to obtain 95 BC1 F1 seeds for each recurrent-donor combination.The BC1 F1 seeds w ere planted in boxes in a greenhouse.Tw o w eeks after planting leaf samples w ere collected from each plant and sent to LGC Geno mics for genotyping w ith the 184 SNPs (supplemental Appendix 1).This allow ed selection in each population of the BC1 F1 individual plants that w ere heterozygous for SNPs associated w ith dr ought tolerance, Striga and/or nematode resistance (foreground SNPs) and carried as many recurrent-parent alleles as possible at other SNP loci (background SNPs) (supplemental Appendix 4).
The selected BC1 F1 individual plants w ere backcrossed w ith Moussa Local to obtain 95 BC2 F1 individuals that w ere SNPgenotyped.In the BC2 F1 generation, the individual plants that w ere heterozygous for foreground SNPs and carried as many recurrentparent alleles as possible at background SNPs w ere identified.Alleles A and B are designated for Moussa local and IT93K-503-1 or IT97K-499-35, respectively (supplemental, Appendix 2 and 3).
In the next cycle, each of the selected BC2 F1 individual plants was backcrossed to Moussa Local to create BC3 F1 lines.Four BC3 F1 individuals from the cross Moussa Local/IT97K-499-35 and three BC3 F1 individuals from the cross Moussa Local/IT93K-503-1 were selfed to obtain about forty BC3 F2 seeds per line.Seed from BC3 F2 w ere used for morphological character ization of the families and y ield performance estimation.Ten entries ( 6 MA BC lines and 4 controls) w ere planted us ing a randomized complete block design (RCBD) w ith tw o replications in tw o water regimes -w ater-stressed (WS) and w ell-w atered (WW).The parents used in the introgression process (IT97K-499-35, IT93K-503-1, and Moussa Local) and one know n drought tolerant variety (Gorom Local) w ere used as checks.The trial w as conducted during the off -season in 2014 from April to June under a drip-irrigation system and striga naturally infested field at the Kamboinsé Research Station, near Ouagadougou, Bur kina Faso.

QTLs introgression
Genotyping of the BC 1 F 1 identified the plant named M503_BC1F1_31 carrying the donor IT93K-503-1 alleles for yield under drought, Striga and nematode resistance, and about 67% of recurrent parent Moussa Local alleles at background markers.In the cross from Moussa and IT97K499-35, the plant named M499_B C1F1_04 carried the donor IT97K-499-35 alleles for yield under drought and Striga resistance, and about 70% of Moussa Local alleles at background markers.In addition, some other BC M499_BC2F1_48P93, M499_BC2F1_48P85, and M499_BC2F1_4P67) were backcrossed to Moussa Local to generat e the BC 3 F 1 families.A total of six families derived from the two donors were retained and selfed (fiveM499_BC3F2s and oneM503_BC3F2s) to increase seed for further studies.

Morphological characterization of the MABC selected lines
Seed from BC 3 F 2 were not enough to undertake a multilocation trial, so they were used for morphological characterization of t he families in a single site yield trial.Table 1 shows the morphological characteristics of the selected families and t he recurrent parent.Figure 1 also shows the plant type or growt h habit of the lines and their dry pods form and color in comparison to Moussa Local.

Yield performance of the MABC selected lines
The yields of the ten lines per water regime are represented in Figure 2. The yields ranged between 287.2 and 1184.70Kg ha -1 in the water-stressed environment, while in the well-watered environment the yields were higher, ranging from 272.3 to 1771.0 Kg ha -1 .Three BC3F2 families (M499_BC3F3_48P85, M499_BC3F3_4P67, and M499_B C3F3_48P90) yielded better than all parents and the local control Gorom Local.All BC3F2 families appeared to perform better than the recurrent parent Moussa Local under limited wat er conditions.

DISCUSSION
This study involving MABC methodology using SNPs breaks new ground for cowpea breeding, particularly in selection for drought tolerance.The methodology enabled rapid rec overy of the recurrent-parent (Moussa Loc al) background (up to 97% ) with only two backcross cycles (BC 2 ) by using ninety-five individuals for each set of backcrosses in the BC 1 generation.This expedient    2013) that revealed a percentage of recovery of 98% at BC3 with a number of 100 individuals selected at BC 2 .
In the present study, several donor loci (yield under drought, stay-green, Striga resistance and root-knot nematode resistance) were introgressed simultaneously.This decreases the chance to identify a line carrying all donor alleles and high Moussa background and therefore, limits the number of offspring to be selected in the subsequent generations.Sebolt et al. (2000) also reported that the rate of success decreases when large numbers of QTLs are targeted for introgression; by using MABC for two QTLs for seed protein content in soybean introgression, they eventually found that only one QTL was confirmed in BC3F4:5.Compared to MABC, conventional backcross breeding, however, requires a much larger backcross population of 500 or more plants to be produced to ensure that there are sufficient plants for background selection after the foreground and recombinant selection have been performed.During this process, unless breeders screen the material to identify those that are carrying the gene of interest, they may need to conduct 'blind crossing' to the recurrent parent.In such conditions conventional backcrossing can be extremely time-consuming and inefficient.By using a combination trait-flanking markers and evenly distribut ed markers across the recurrent parent genome, effective introgression c an be done to avoid linkage drag and t he use of large numbers of individuals.
In Burkina Faso, most of the cowpea landraces have a prostrate growth habit and are susceptible to Striga and are often grown with cereal crops like sorghum and millet.
The prostrat e growth habit allows soil conservation and maintenance of soil moisture by the cover of the vines.These morphological characteristics in the selected advanced BC lines are in accordance with the expectations of recovering the recurrent parent background (Figure 1).Moussa Loc al, a farmer -preferred landrace has some purple pods which remain purple even for dry pods.The six selected lines had the purple green and dry pod color character confirming that they recovered this character from t he recurrent parent.The prostrate (spreading) growth habit of Moussa Local was also found in the selected MABC lines.These result s, therefore, confirmed the molecular results that showed a high level of recovery of the Moussa Loc al rec urrent parent plant type.The same trend was observed for Striga resistance.Only one line (M499_B C3F3_48P93) that had no Striga donor allele had Striga emergence in the well-wat ered environment.The Striga-resistant checks did not emerge Striga confirming their resistance while Moussa local had emerged Striga confirming its susceptibility to Striga.This result also confirms that the lines selected based on the presence of the Striga gene through the MABC introgression are effective in controlling Striga.
Yield is by far the most important criterion for varietal selection by A frican farmers (Tignegre, 2010;Some, 2012;Traore, 2013).Promising results from the preliminary yield performance trial indicat ed that three lines yielded better than the parents and the droughttolerant control Gorom Local (Figure 2).In addition, the general performance of these lines reached the potential yield of the recently released varieties in Burkina Faso which is around 1.5 t.ha -1 (Ouedraogo et al., 2012).The low yields of certain lines could be attributed to the fact that there were drought spells due to water shortages during the growing period at Kamboinsé.However, other authors have reported in maize three QTLs for two traits (earliness and yield) were int rogressed between maize elite lines with MAB C but the results were influenced by the function of other genes cont rolling the traits (Bouchez et al., 2002).
In the domain of molecular breeding, a lot of conventional breeding methods have been associated with markers to design a large number a marker-aided selection methods.Some examples are marker-assisted selection (MAS), marker-assisted recurrent selection (MARS), marker-assisted backcrossing (MAB C), and ultimately genomic selection (GS).Among t he molecular breeding methods, MABC has been the most widely and successfully used in plant breeding up t o date.Markerassisted backcrossing (MABC) is an effective method for developing improved versions of widely cultivat ed varieties, also referred to as Mega varieties (Neeraja et al., 2007).It has been applied to different types of traits (e.g.disease/pest resistance, drought tolerance and quality) in many species, e.g.rice, wheat, maize, barley, pear millet, soybean, tomato, etc. (Collard et al., 2005;Dwivedi et al., 2007;Xu, 2010).

Conclusion
The improvement of cowpea varieties by adding traits through MABC is now becoming a significant advanc ement in Africa, and work is continuing to transfer drought tolerance QTL, Striga resistance, aphid and nematode resistance among other t raits into a range of economic ally important parental lines at INE RA.The use of the marker technology tested in this study has allowed rapid recovery of the background of a farmer preferred widely grown cowpea landrace.The morphological characteristics of the improved lines developed in this study indicated that they could be good candidates for production under intercropping in farmer fields.Until now, no Striga resistant variety combining preferences has been proposed by INE RA for intercropping.Since Moussa Local is used in intercropping, these improved lines hold much promise for performance under this production system.From this study three of the six MABC-improved lines are most promising based on good yield potential and their resistance to Striga.These three lines (M499_B C3F3_48P85, M499_B C3F3_4P 67, and M499_BC3F3_48P90) are being seed-increased for multi-location trials to measure their performance and ability to withstand drought events and Striga attack.

Supplemental
Appendix 1: SNP ID and sequences used for MABC selection

Figure 1 .
Figure 1.A-Plant grow th habit of selected MA BC lines field-grow n during the offseason w ith drip-irrigation; B-Dry pod shape and color of selected MABC lines compared to Moussa Local (recurrent parent).

Figure 2 .
Figure 2. Yield perfor mance of selected BC3 F2families, their parents and a local control under w ellw atered and limited-w ater conditions.Values are the yield mean (kg/ha) of tw o replications.

Table 1 .
Morphological characteristics of MABC selected lines and their recurrent parent RP: recurrent parent.