Introgression of anthracnose resistance Co-4 gene in elite French bean (Phaseolus vulgaris L.) background using linked molecular marker SY20

French bean is one of the most important staple foods in many parts of the world. Among several bacterial, fungal and viral diseases, anthracnose caused by Colletotrichum lindemuthianum is the most widespread and severe disease of French bean. Therefore, the present study was undertaken as prebreeding effort with the objective of introgression of important bean anthracnose resistance gene Co-4 in elite cultivar. Hybridization between resistant genotype, TO having bean anthracnose resistance gene Co-4 and elite cultivar Arka Komal was done. Cross Arka Komal × TO was further advanced to generate, F1, BC1F1, BC2F1 and BC2F2 plants. Testing of hybridity of the resultant F1, BC1F1, BC2F1 and BC2F2 plants/pods using gene linked SCAR marker SY20 resulted in identification of 46 gene-positive BC2F2. Further foreground selection revealed lack of segregation for the target gene Co-4 in 16 progenies. Screening of these 16 homozygous progenies (Co-4 gene) following detached pod and germinated seed dip methods using race 3 of C. lindemuthianum validated Co-4 imparted resistance in all the 16 BC2F3 progenies. Agronomic evaluation of 16 Co-4 gene-positive BC2F4 progenies for 8 qualitative traits and 9 quantitative traits led to identification of AKTO 4, AKTO 5, AKTO 7 and AKTO 43 progenies as having better elite background. These can either be used as donors of resistance gene for further introgression and gene pyramiding or can further be advanced following backcrossing to develop essentially derived variety of elite parent, Arka Komal.


Introduction
French bean belongs to order Fabales and family Fabaceae, sub family Papilionideae, tribe Phaseoleae, sub-tribe Phaseolionae and genus Phaseolus. French bean is an autogamous diploid (2n = 2x = 22) species with a comparatively small genome 587 mbp (Bennett and Leitch 1995;Broughton et al. 2003) and genetic consensus map is structured into 11 chromosomes (Nodari et al. 1993;Freyre et al. 1998;Pedrosa-Harand et al. 2008). Beans comprises of two major eco-geographical gene pools, Middle America and the Andes (Kwak and Gepts 2009). French bean is a rich and low-cost source of protein, ber and other important minerals, i.e. iron, zinc, copper, phosphorus, potassium, magnesium, calcium and many vitamins (Limongelli et al. 1996; Kaur et al. 2009; Khetan et al. 2015). Therefore, the crop is considered an economically, nutritionally and socially important (Broughton et  Several bacterial, fungal and viral diseases attack French bean and among these anthracnose caused by fungus Colletotrichum lindemuthianum is one of the most widespread and severe. This disease can cause yield losses of up to 100% when infected seed is sown in areas with high relative humidity and mild temperatures range from 18 to 24 ˚C (Peloso 1992). Majority of the recommended and locally adapted bean cultivars in Himachal Pradesh are susceptible to different races of pathogen (Sharma et al. 1999). The most prevalent races in the region are race 3, 115, 513, 515, 529, 537, 615, 631, 775 and 935 (Pathania et al. 2006).
Studies on race spectrum of bean anthracnose have revealed presence of 27 races in all from North Western Himalayan region, out of which 19 are reported to be present in Himachal Pradesh (Sharma et al. 2019) Both cultural management and use of fungicides can be followed to control the disease. However, chemical control is found to be expensive and generation of pathogen free seeds is often di cult in developing countries. So, the use of resistant genotypes emerges to be the most biologically safe and cost-effective management strategy (Pastor-Corrales and Tu 1989). A large number of studies on genetics of resistance to anthracnose have revealed it to be governed by 26 anthracnose resistance loci identi ed and denoted by the symbol Co including Co-1 to Co-7, co-8, Co-9 to Co-18, Co-u to Co-z ( farmers of the state however most of these are susceptible to many prevalent races of bean anthracnose. Arka Komal is such preferred bush type French bean variety grown by the farmers of Himachal Pradesh and its adjoining hilly and plain areas which are highly susceptible to this disease. Thus there is need to develop French bean variety having resistance to bean anthracnose to counter threats of this disease on bean production. Therefore, the present study is proposed with the objectives of introgression of important bean anthracnose resistance gene Co-4 in elite cultivar and to assess the derivatives for validation of imparted resistance. The study will result in identi cation of anthracnose resistant derivatives which can further be advanced to isolate derived variety (ies) and used as prebreeding material in bean anthracnose breeding programme.

Plant material
The French bean genotype TO (Co-4) was used as resistance donor against bean anthracnose, whereas French bean Arka Komal was used as recipient parent.
Hybridisation and backcrossing to generate F 1, BC 1 F 1 and BC 2 F 1 seeds The hybridization was attempted between bean anthracnose resistance gene donor genotype TO and elite cultivar Arka Komal gene donor as male parent. The F 1 progenies (Arka Komal × TO) were then backcrossed with respective elite parent using F 1 as male parent. The process of hybridization involved emasculation of the owers and arti cial pollination of emasculated owers next morning.
The data on total number of owers emasculated and pollinated and total number of pods set were collected cross-wise.
Crossability was expressed as the per cent pod set in hand pollinated owers and was calculated as follows: Crossability (%) = (Total number of pods set / Total number of owers pollinated) × 100 Marker assisted foreground selection The DNA for marker analysis was extracted by CTAB method (Murray and Thompson, 1980). The SCAR marker SY20 reported to be linked to the targeted bean anthracnose resistance gene Co-4 was used to identify the plants carrying the resistance gene in F 1 , BC 1 F 1 , BC 2 F 1 , BC 2 F 2 and BC 2 F 3 progenies.
Isolation, puri cation and quanti cation of the plant genomic DNA Genomic DNA of plants of parents and various generations were isolated from young leaves using CTAB method (Murray and Thompson 1980). The extracted DNA samples were loaded on 1% agarose gel (1gm/100ml 1X TAE Buffer) and run at 90 V for 30 mins to ascertain the quality and quantity of DNA.
PCR ampli cation DNA samples were used for PCR ampli cation by using SY20 SCAR primer (F: AGCCGTGGAAGGTTGTCAT, Tm -56.9˚C; R: CCGTGGAAACAACACACAAT, Tm -53.9˚C). For ampli cation of genomic DNA, a reaction mixture was prepared using PCR water, DNA (15-25 ng/µl), 10X buffer (10 mM), dNTP mix (0.5 mM each of dATP, dGTP, dCTP, dTTP), forward primer (5 pM), reverse primer (5 pM) and Taq polymerase (3 U/µl). The ampli cation was carried out in Veriti 96 wells Thermal Cycler (Applied Biosystem, CA, USA) following PCR ampli cation pro le for Co-4. The PCR cycle for SY20 scar marker consisted of 2 min at 94˚C; followed by 30 cycles of 30 s at 94˚C, 1 min at 59.2˚C and 1.5 min at 72˚C; a 5 min extension at 72˚C; and a hold at 4˚C.

Analysis of PCR products
For analysis of PCR products, 2 % agarose gel was prepared in 1X Tris-Acetate-EDTA and ethidium bromide (0.5µg/ml) was added to gel solution before setting the gel on casting plate. The PCR products (10 µl) along with 2µl loading dye (0.25 % bromophenol blue and 40 % sucrose) were loaded in the gel inside the electrophoresis tank along with a 100 bp DNA ladder and resolved on 2 % agarose gel. Electrophoresis was carried out at 100-120 V for 90 mins and nally the gel was visualised and photographed using the Gel-Documentation Unit (Labnet, ENDURO TM GDS, Aplegen). Presence/absence of appropriate size product of the marker SY20 (830 bp) linked to the anthracnose resistant gene, Co-4 was recorded to infer the presence of Co-4 gene in the parents and progenies.

Validation of anthracnose resistance
Cultures of C. lindemuthianum race-3 showing virulence on susceptible parent and avirulence on Co-4 gene harbouring resistant parent To, were used for screening and validation of Co-4 imparted anthracnose resistance in homozygous selected lines harbouring resistance genes. Validation of resistance was done following detached pod and germinated seed dip method.
Screening by detached pod method was repeated twice to con rm the disease reactions. Infection type on inoculated pods was evaluated for disease severity by following the rating scale of 0-9 (Mayee and Dattar 1986)  Pods scoring reaction types of 0, 1 and 3 were graded as resistant while those scoring 5, 7 and 9 were graded as susceptible.
In seed dip method the reactions of inoculated seedlings were scored after six and twelve days of inoculation following 0-5 scale The shape of pods of all plants of each line was observed visually and categorized on majority basis as curved, moderately curved, slightly curved and straight.

Pod colour at picking stage
The pod colour at picking stage was observed visually and categorized on majority basis as green.

Stem colour
The stem colour was observed visually and categorized on majority basis as green.

Seed coat pattern
The seed coat pattern of the seeds was characterized as mottled and absent on each homozygous progeny/line.

Seed coat colour
The seed coat colour of the seeds was observed visually and categorized as different ranges from greyish white to brown, greyish white to dark brown, greyish white to light brown, greyish white to beige, greyish white to light beige, greyish white to golden brown, greyish white to pinkish brown and greyish white.

Presence of colour on around of hilum
The presence and absence of colour on around of hilum was observed visually on each homozygous derivative line along with parents.

Brilliance of seeds
The brilliance of seeds was characterized as medium and shiny on each homozygous progeny/line along with parents.

Pod length (cm)
Length of the ten randomly chosen green pods picked from 5 selected plants was measured and average recorded in cm for each line in all replications.
Green pod yield/plant (g) The green pod yield obtained from the 5 randomly selected plants under different pickings was summed and average recorded in grams (g) for each line in all replications.

Statistical analysis
The data collected on 9 quantitative traits were subjected to analysis of variance (ANOVA) following standard statistical procedure following Panse and Sukhatme (1984)  The entries/genotypes mean sum of squares were tested against error mean squares by "F" test at (g-1) and (r-1) × (g-1) degree of freedom at P≤0.05 for statistical signi cance of differences among genotypes/entries. Based on the above ANOVA, the following statistical parameters were calculated for inference of the data and its interpretations: The seeds of gene-positive plants identi ed above were harvested as single plants and advanced as plant to row progenies to develop BC 2 F 3 plants. Ten seeds each of 46 BC 2 F 3 plant progenies were sown as rows (Progeny 1 to 46) in the cage house. The information on total number of seeds harvested from each BC 2 F 2 gene-positive plant, number of seeds sown, germinated and segregation status for the target gene in each plant to row progeny of cross is presented in Table 1.
A total of 1259 BC 2 F 3 seeds were harvested from 46 selected gene-positive BC 2 F 2 plants, out of which 9-10 seeds of each progeny were sown as single rows. Validation of Co-4 imparted anthracnose resistance by using cultures of race 3 of C. lindemuthianum Validation of resistance in 16 selected homozygous lines for Co-4 gene was done by following detached pod and germinated seed dip method (repeated twice). Culture of C. lindemuthianum race-3 showing virulence on susceptible parent and known to be avirulent on Co-4 gene was used for screening and validation of anthracnose resistance of 16 selected homozygous lines carrying Co-4 gene as inferred by ampli cation of gene linked SCAR marker. Results of screening using both methods are presented in Table 2 and disease symptoms/reactions shown in Figure 7 (detached pod method) and Figure 8 (germinated seed dip method).
Screening for bean anthracnose using detached pod method revealed a disease score of '5' in the recipient parent, Arka Komal, whereas in resistant donor TO a disease score of '0' was recorded. All the 16 BC 2 F 3 progenies inferred to be homozygous for the target gene Co-4, recorded a disease score of '0' revealing there by resistant reaction of donor and 16 backcross progenies. Germinated seed dip method con rmed these results wherein also disease score of '0' was recorded in donor parent TO and 16 backcross progenies. These results clearly validated the presence of anthracnose resistance gene Co-4 selected through linked marker SY20 and Co-4 gene imparted resistance to race 3 of C. lindemuthianum in all 16 selected progenies. progenies, AKTO 5 and AKTO 7 were graded as moderately curved and some pods in AKTO 7 had curved pods along with moderately curved.
Pod colour at physiological maturity stage was noticed visually and pod colour of parent, Arka Komal and TO was observed green.
All 16 Co-4 gene-positive progenies exhibited green pod colour at physiological maturity stage. Stem colour of parents and backcross progenies revealed that Arka Komal and donor, parent TO (Co-4) has green stem colour. All 16 Co-4 gene-positive backcross progenies also exhibited green stem colour. There is absence of any seed coat pattern in the seeds of recipient parent, Arka Komal whereas seed coat pattern of the donor parent, TO was observed to be mottled. All 16 Co-4 gene-positive back cross progenies were observed to have mottled seed coat pattern ranging from sparse to dense brown in colour. Seed coat colour of recipient parent, Arka Komal was observed grayish white to brown and donor parents, TO were noticed grayish white to beige. Seed coat colour of Co-4 gene-positive back cross progenies exhibited a large variation ranging from grayish white to beige, grayish white to light beige, grayish white to dark brown, grayish white to golden brown, grayish white to pinkish brown and golden brown (

Discussion
The crossability relationships between and within Phaseolus species have been extensively studied and reviewed (Smartt 1990;Debouck and Smartt 1995). Generally, there are strong barriers for interspeci c hybridisation in Phaseolus, but between each cultigens and its wild ancestral type, almost full genetic compatibility exists (Mumba and Galwey 1998). Mumba and Galwey (1998) in their exhaustive study on cross compatibility between gene pools and evolutionary groups in Phaseolus vulgaris L. has clearly demonstrated that there is no general consistent tendency for pollinations between genotypes within a gene pool to be more successful than those between gene pools. In evolutionary groups (wild, landrace and bred), the crosses among landrace and bred genotypes, those within evolutionary classes had higher success rates than those between classes (47.2% vs. 38.9%).
In the present study, the extent of crossability recorded (7.69%) can easily be considered as comparatively low as the parent of the present study belong to the same evolutionary group i.e. bred genotypes should have shown higher compatibility as reported by the aforementioned studies. However, the e ciency of crosssing depends on the fact that the stigma and pollen should be in very close contact as long as possible to ensure fertilization, as occurs with in the keel in natural sel ng. Low cross compatibility in the present case can probably be ascribed to comparatively unfavorable factors for hybridization mentioned above under cage house as well as to the limited skill of person handling hybridisation. Similarly experience in manual hybridisation is likely to enhance the success rate of hybridisation programmes.
Overall in cross (AK × TO), the observed segregation ratio of gene-positive and gene-negative plants was 67.65% and 32.35% as against the expected perfect t of 75% and 25%, respectively. Such minor deviation can be a result of small sample size of population, differential seed development as well as a result of selection of seeds as only well lled seeds were advanced in the present case.
Marker assisted selection (MAS) and marker assisted backcross breeding (MABC) have received much attention as a viable method for crop improvement. In the case of disease resistance genes, molecular markers linked to genes of interest can be used to accumulate R genes in a single cultivar without laborious disease screening (Miklas et al. 2006). There is need to develop common bean varieties having resistance to bean anthracnose to counter threats of this disease on bean production. Chemical control is found to be expensive and generation of pathogen free seeds is often di cult in developing countries. So, the use of resistant genotypes appears to be the most biologically safe and cost-effective management strategy (Pastor-Corrales and Tu 1989).
In common beans, many earlier studies have also reported successful use of linked molecular markers for the selection of different bean anthracnose resistance genes in early as well as advanced segregating material. RAPD markers, OPY20 830C (0.0 cM) and OPC08 900C (9.7 cM) linked to the Co-4 gene have been identi ed by Arruda et al. (2000) in the coupling phase. It has been reported by them that the selection e ciency for resistant and susceptible plants was 100 per cent with RAPD marker OPY20 830C due to its close linkage to locus Co-4 (0.0 ± 1.1 cM) and hence this marker can be used for screening gene harbouring plants in advanced generation (F 6 or later) lines that are nearly homozygous. The SCAR marker, SY20 used in the present study for foreground selection of Co-4 gene has been developed from this RAPD marker (Queiroz et al. (2004) and therefore is also expected to be equally e cient in indirect selection for the gene, Co tested four SCAR markers in the present work (SH18 and SBB-14 for Co-4 2 , SAB-03 for Co-5, and SY-20 for Co-4), have shown to be useful for marker assisted selection of the target resistant genes. They were found speci c for the linked loci. SH18 was shown to be speci cally linked to Co-4 2 and some other did not discriminate alleles from the same locus.
One of the major constraints during breeding for disease resistance is that donor lines are carrier of undesirable attributes that subsequently lead to development of poor performing progenies from crosses of elite parents with gene donors (Hospital 2005). Various approaches are followed to reduce this junk and linkage drag including marker-assisted backcross breeding (MABC) with foreground and accompanying background selection to restore the elite background (Fukuoka et al. 2009). Under such situations, one of the alternative approaches could be pre breeding efforts to bring such important disease resistance genes into comparatively better and/or elite backgrounds, and to use products of such breeding efforts as donors of the genes rather than the original inferior donors.
Studies on race spectrum of bean anthracnose have revealed presence of 27 races in all from North Western Himalayan region, out of which 19 are reported to be present in Himachal Pradesh (Sharma et al. 2019). Apart from race 3, Co-4 gene is reported to impart resistance against most of the other Indian races including 115, 513, 515, 529, 537, 615 and 631 (Pathania et al. 2006). Out of these 8 races, four new races (115, 537, 615 and 631) have been reported for the rst time from Himachal Pradesh, India which had not been reported earlier from the other regions of the world (Sharma et al. 2007). Therefore, deployment of the gene Co-4 is more relevant in this region as the lines developed under this study harbouring Co-4 have potential to impart resistance against 8 prevalent races, four of which are new and speci c to the region. Therefore, deployment of the gene Co-4 is most relevant to achieve durable and e cient resistance against bean anthracnose.
In literature, various methods of arti cial disease screening for anthracnose resistance are mentioned including seedling inoculation, germinated seed dip inoculation, detached pod and detached leaf methods (Tu 1986). Of these detached leaf and pod method have been reported to be the best by Bigirimana and Hofte (2001). Different methods have associated advantages and disadvantages. In detached leaf/pod method, spores are uniformly distributed over the leaf/pod area which provides accuracy and promptness for screening because disease symptoms appear uniformly on leaf/pod area. However, the results of the present study clearly demonstrated equal e ciency and accuracy of both, detached pod and germinated seed dip methods as similar disease reactions were obtained on test cultures using both methods.
As discussed above, SCAR marker SY20 linked to gene, Co-4 is reported to be used successfully to select gene-positive progenies in different bean breeding programmes throughout the world (Queiroz et al. 2004 andKelly 2001). As far accuracy and e ciency of markers is concerned, the present study has clearly indicated the cent per cent accuracy and e ciency of SCAR markers SY20 in selecting the gene, Co-4 as indicated by the validation of imparted resistance in all 16 progenies. This is easily understandable in case of marker SY20 that is known to be tightly linked with the gene Co-4 gene has been developed from OPY20 830C RAPD marker at a distance of 0.0 ± 1.1 cM (Queiroz et al. (2004) and therefore is also expected to be equally e cient in indirect selection for the gene, Co-4.
In French bean pod shape is gaining more importance as an important agro-morphological attribute because of its role in post harvest processing. Straight pods are preferred more for easy handling and transport. Hence four Co-4 gene-positive progenies exhibiting straighter pods can be likely used as donor of traits in addition to resistance gene, Co-4. BC 2 F 4 progenies AKTO 4, AKTO 5, AKTO 7 and AKTO 43 of cross were identi ed as having better elite background along with the target gene can be potentially used as donor of various traits in addition to bean anthracnose resistance gene, Co-4 as the likelihood of such parents contributing poor traits would be far less than the original donors and associated linkage drag can also be minimized.

Conclusion
The present study revealed the possibility of hybridisation and clearly validated the use of SCAR marker SY20 for foreground selection in backcross breeding and tracking the genes in segregating populations of beans in selecting the gene Co-4. Comparable/better performance for some agro-horticultural traits revealed elite background of the identi ed stable homozygous derivatives (BC 2 F 4 ) for gene, Co-4 indicating success of backcross programme undertaken in the current study. The identi ed derivatives of cross can be further advanced using backcrossing to further improve the background and/ used as donors of resistance genes in the future bean breeding programmes.     Table 5 Performance of the parents and 16 Co-4 gene-positive BC 2 F 4 progenies for 9 quantitative agro-horticultural traits