*6.1. Utilization of Pathogen-Free Planting Material*

Fusarium wilt can be spread by infected seeds and plant waste [102]. Using infected propagation material, the pathogen is transferred into productive areas or soils that are pathogen-free. Therefore, the significance of monitoring the health of the item through certification programs under quarantine legislation and phytosanitary inspection should be taken in to consideration. The right choice of planting site is aided using *F. oxysporum* spp.-free planting material in non-infested soils [102].

### *6.2. Chemical Control*

Chemical control is one of the finest disease management strategies for diseases that are spread through soil. FW can be controlled using organic chemical methyl bromide, which is a very effective fumigant. This chemical was used by Animisha et al. [100] to control FW. In addition to this, some popular fumigants, including dazomet, chloropicrin, carbendazim, and 1,3-dichloropropene, were also employed to combat FW in pea and chickpea, respectively [101].

#### *6.3. Biological Control*

An integrated disease management strategy can easily include biological control and plant resistance as a cost-efficient and environmentally beneficial method of disease control [102]. An effective cure for chickpea wilt disease has been demonstrated using an arbuscular mycorrhizal consortium to control the biological processes of Fusarium wilt [103]. Numerous biocontrol agents have been used effectively and have led to a significant decrease in both pathogenic fungal growth in vitro and disease development in plants [104]. These bacteria and fungi include non-pathogenic and non-host Fusarium species [105]. The *Pseudomonas fluoresces* formulation treatment has increased chickpea production in the field and can be applied as a seed treatment to prevent chickpea wilt. Additionally, Fravel et al. [106] linked higher plant defensive responses to root colonization by the non-pathogenic strain of *Fusarium* spp. with disease reduction [107]. In a study, it was discovered that pre-treating chickpea seedlings with *Rhizobium* isolates before subjecting them to *FOC* increased the levels of total phenolics, constitutive is flavonoids, for mononetin, and biochanin [108]. The protection of chickpea against Fusarium wilt by non-pathogenic and non-host Fusarium species has been linked to the induction of the phytoalexins medicarpin and maackiain, as well as the related isoflavones formononetin and biochanin A [109].

#### *6.4. Cultural Control*

Fusarium wilt disease in numerous crops was successfully controlled using the soil solarization method [110]. The heat produced by solarization may not kill the pathogen, but it may weaken it, reducing its host's sensitivity and increasing its susceptibility to assault by other soil microflora members [111]. The risk of disease in the following crop could be reduced by clearing away the debris from a field that has been afflicted by Fusarium wilt and igniting or burning it to destroy the *FOC* chlamydospores. Temperature has a big impact on chickpea's ability to resist Fusarium wilt. When there is a rise in temperature of 2–3 ◦C, different races of *Fusarium oxysporum* f. sp. ciceris (*FOC*) become more vulnerable to pathogens [112].

According to an investigation by Orr and Nelson [113], the Fusarium wilt pathogen in chickpea can live in the soil for up to 6 years, and 3 years of crop rotation is ineffective in lowering the incidence of the disease. In a 1998 study in southern Spain, Navas-Cortes found that planting date had the greatest impact on epidemic development. Sowing chickpea crops later in the year, from early spring to early winter, can slow the spread of Fusarium wilt epidemics and boost chickpea seed production [112].

#### *6.5. Use of Resistant Cultivars*

The most practical and cost-effective technique for controlling Fusarium wilt is the use of resistant cultivars. However, several factors that affect disease resistance, such as genetic and pathogenic variability, the evolution of the pathogen, the availability of resistance sources, the co-infection of plants with other pathogens, genetics, and the penetrance of resistance (i.e., reduced expression as a result of the interaction between host genotype and inoculum load, temperature, and seedling age), etc., can seriously limit its use and effectiveness [112,113]. A crucial element of the integrated disease management (IDM) program is the use of resistant chickpea cultivars and additive or synergistic combinations of biotic, cultural, and chemical control strategies [112]. The use of resistant cultivars has been restricted because certain novel materials have undesirable agronomic characteristics. Furthermore, the effectiveness and widespread use of current resistant cultivars may be constrained by the considerable pathogenic diversity of *FOC* populations [114].

Recent years have seen significant challenges in achieving the desired yield of chickpea due to various factors. In most chickpea-growing regions, studying different stressors is important [90]. Future work should therefore concentrate on creating cultivars that are multi-stress-resilient. A thorough comprehension of significant pressures and the genetics of resistance ought to result in more methodical methods of resistance breeding. It is important to breed wild Cicer species for resistance because they have a lot of potential [99].

#### **7. Advanced Breeding Techniques**

The study of an organism's entire genome is referred to as genomics. Recombinant DNA, DNA sequencing techniques, and bioinformatics are all combined in genomics to sequence, assemble, and analyze the structure and function of genomes [115]. Genomic science is the study of how genes and genetic data are structured inside the genome, the procedures for gathering and evaluating these data, and how this organization influences their biological usefulness. The three key fields of genomic biology are structural, comparative, and functional (Figure 3) genomics [116]. With the goal of understanding evolutionary linkages and how genes and genomes function to produce complex phenotypes, such as gene regulation and environmental signaling, genomics is a branch that aids in comprehending the sequencing of genes and genomes [117].

**Figure 3.** Omics approaches and their role in chickpea breeding.

### *7.1. Marker Technology*

There are three types of markers generally used in crop improvement programs including phenotypic, biochemical, and molecular markers [118]. Among these markers, molecular markers are more authentic due to their neutral behavior in different environmental conditions. Nucleotide sequences make up molecular markers, and the variation in nucleotide sequences among different individuals makes it possible to study these sequences [119,120]. The use of molecular markers that are closely related to the genes or QTLs controlling Fusarium wilt resistance allows for quicker and more accurate breeding. Although they are created through insertion, deletion, point mutations, duplication, and translocation, these polymorphisms are not always connected to the activity of the genes [121,122].

The genetic marker is a gene or DNA sequence with a known chromosome location that regulates a certain gene or characteristic. Genetic markers are closely related to the target gene and act as warning indications or flags [118]. Meanwhile, in contemporary genetics, genetic polymorphism describes the relative variation in the genetic loci of the genome. Genetic markers can be used to aid in the study of heredity and variation. Recent advances in molecular breeding, including the use of PCR-based techniques, such as simple sequence repeats (SSRs), insertion/deletion mutations (Indels), single-nucleotide repeats (SNPs), genomic sequencing (GS), genotype by sequencing (GBS), etc., have been widely used in crop improvement programs worldwide [119].

In contrast to multi-locus markers, including random amplified polymorphic DNA (RAPD), arbitrarily primed polymerase chain reaction (AP-PCR), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and sequence-specific amplification polymorphism (S-SAP) markers [120], the single-locus markers—including fragment length polymorphisms (RFLPs), variable number tandem repeats (VNTRs), simple sequence length polymorphisms (SSLPs), sequence-tagged microsatellite sites (STMSs), simple sequence repeats (SSRs), sequence tagged sites (STSs), single-nucleotide polymorphisms (SNPs), cleaved amplified polymorphic sequences (CAPSs) and sequence-characterized amplified regions (SCARs)—are frequently used in plant breeding in a variety of studies. In modern plant breeding, single-locus markers are used for various purposes, including germplasm characterization and protection, gene tagging, genome mapping, linkage map construction and analysis, evolution studies, parental selection, F1 hybrid testing, genetic purity testing of seeds, genes, QTL mapping, etc. [121,122]. Employing marker loci that are strongly connected to vital genes that regulate features with economic relevance, such as disease resistance, male sterility, self-incompatibility, and seed qualities (including form, size, color, and texture) can help in selection.

#### 7.1.1. Molecular Markers and FW Resistance in Chickpea

The identification and creation of genetic maps of the segregating population are breeder's top priorities. Utilizing molecular markers for labeling traits and site-specific genes of interest, chickpea genetic maps have been created [123]. Using isozymes from the F2 population resulting from interspecific crosses, the first maps were produced [124]. Numerous studies have discovered genes that influence floral color, wilt resistance (Fusarium), double pods, and growth behavior [123,125]. Higher numbers of maps connected to features were derived using multiple markers, crosses from *C. reticulatum*, and other techniques. Microsatellite markers, however, were used to create populations from interspecific crosses, which take advantage of more genetic variations among chickpea genotypes [126]. The first transcriptome study of the chickpea genome was finished after the development of next-generation sequencing [127]. With the development of transcriptome information, detailed genetic maps were created using large-scale molecular markers [128–130]. The genetic population utilized to map and find QTLs in the chickpea genome may benefit from having access to draught genome sequencing in the Desi and Kabuli types [131]. Omics methods gathered genomic data and sparked the development of tightly connected QTLs in molecular markers [132].

The diseases for which significant resistance genes have been backcrossed into elite cultivars are the ones for which MAS in plant breeding is most effective [133]. Chickpea provides some evidence of the application of MAS to facilitate efficient and accurate breeding. The SSR markers, namely, TR19, TA194, and TA660, which were discovered to be polymorphic between the parental lines, have already been used for foreground selection via marker-assisted backcrossing in order to introduce *FOC*1 in a superior chickpea cultivar [134]. As part of marker-assisted introgression, the SSR markers TA110 and TA37 in chickpea LG2 were also used to introduce *FOC*-2 into the background of a superior cultivar [135]. To develop virtually isogenic lines with disease resistance, TA59, one of the several markers discovered to flank the *FOC* race 5 resistance gene, was used [136].

The use of molecular markers is an essential method for classifying, characterizing, and screening infections and diseases. To categorize and filter fungi, internal transcribed spacer (ITS) markers are often used. Even though information on pathogen variety is required to comprehend pathophysiology and development for management strategies, SSR markers are employed in unique backcross generation to aid in the selection against Fusarium resistance. The importance of resistant molecular markers in identifying diseasecausing genes and resistance mechanisms has been acknowledged. Numerous crops have additionally demonstrated a substantial association between microsatellite markers and resistance genes, such as Fusarium wilt resistance genes, in chickpea, and many others.

Initial efforts to map resistance genes using restriction fragment length polymorphism (RFLP), RAPD markers, and isozymes failed. Only modest polymorphism was detected in chickpea using the resistant gene analogue (RGA), ISSR, and RAPD [137]. Nevertheless, *FOC*1 was mapped at 7.0 cm on the same side of the gene using two markers, viz., CS27700 and UBC170550. The resistance genes *FOC*3, *FOC*4, and *FOC*5 were later mapped using ISSR, RAPD, and SSR markers [138].

The first WR gene discovered was H1 against race 1 in chickpeas [139]. Two primers, UBC-170550 and CS-27700, respectively, amplified susceptibility and the DNA region linked to FW resistance [140]. However, after transforming these two markers into allele-specific associated primers (ASAPs), only CS-27700 was shown to be specific to the susceptible allele, whereas the other one (UBC-170550) appeared to be locus-specific. The same RAPD markers were later shown to be connected to the gene controlling race 4 resistance at 9 cm [141,142]. ISSR markers were also applied to tag the WR gene in a population that was inter-specific to the mapping method. The authors discovered two ISSR markers associated with the resistance gene for race 4: UBC-855500 and UBC-8251200.

SSR markers are the preferred markers for plant breeding or for plant breeders owing to their multi-allelic and co-dominant properties [143]. The development of SSR markers has made the application of genomic and transcript databases feasible. Several hundred SSR markers have been developed from genomic DNA libraries [144]. The "ICRISAT Chickpea Microsatellite" (ICCM) markers are a set of 311 distinct SSR markers that were created by Nayak et al. [144] using information from an SSR-enriched genomic library of the chickpea accession ICC 4958. Additionally, SSR markers (ESTs) have been mined using expressed sequence tags [144,145]. Primer pairs were created by Varshney et al. [145] for 177 unique EST-SSR markers, and 3728 SSR markers were found.

Using DNA markers, marker-assisted selection can expedite conventional breeding [146,147]. The resistant genotypes of chickpea that were discovered in this investigation may be employed in breeding programs to breed resistant cultivars. Previously, resistance to *FOC* races 1, 2, and 3 was delivered through genes 3, 2, and 1, respectively. The marker CS27 was first associated with *FOC* 1 at 7.0 cm by Mayer et al. [139], and later, this marker was modified to become an allele-specific related marker (CS27A). The *FOC*2 resistance gene was found at 2.7 cm and 0.2 from the SSR markers H3A12 and TA96. The formerly discovered DNA markers proved useful in establishing relationships to phenotypic data and connections to *FOC* 2 resistance genes. This was accomplished by using molecular markers, such as the ASAP marker (CS27700) and several STMS markers [139,148,149]. Utilizing the primers TA110, TR19, TS82, and CS27, a total of 28 genotypes were screened, and it was

found that these genotypes were strongly related with *FOC* 2 resistance genes [138,150]. Resistance gene analogue, DNA amplification, fingerprinting, and other later-developed chickpea markers demonstrated more polymorphism compared to isozymes, RAPDs, and RFLPs. Nevertheless, the development of polymorphic markers led to substantial advancement in the discovery of STMS markers [151].

#### 7.1.2. Marker-Assisted Breeding

Marker-assisted selection (MAS), among other genomic methods, can significantly improve chickpea breeding programs [152]. How well MAS performs depends on the degree of association between the marker and the gene locus determining the target feature. The positioning of the marker in a genomic area with higher levels of polymorphism and simplicity of interpretation can affect the MAS technique [153]. The main advantage of MAS over traditional selection is the capacity to choose features that are difficult or inconvenient to assess directly, eliminating complicated and time-consuming evaluations. This is true when breeding for disease resistance is performed. By pyramiding different resistance genes in a single genotype, MAS also enables quicker variety release and development [154]. An effective technique for utilizing the potential of genes for agronomic traits is marker-assisted selection [155].

For orphan pulse crops, the success of MAS in cereal crops serves as a model. Many genetic resources have recently been invented and employed in marker-trait association research in pulses [156]. Under the auspices of the Indo-US Agricultural Knowledge Initiative (AKI) program, the Government of India, and the Indian Council of Agricultural Research (ICAR) launched the chickpea genomics initiative program.
