**2.6 Marker assisted characterization of germplasm**

In the twenty-first century, food, water and land are biggest challenges for increasing population. Agricultural activities need to expand, become sustained, and be more adaptive to climate change. To improve sustainability in agricultural systems, new paradigms are required, to explore the genetic potential of the huge although

unfortunately underutilized resources of genetic diversity available for different crops. For breeding of climate-resilient varieties, a better understanding of evolutionary genetic variability is essential. Genetic diversity is the precious wealth for any crop improvement programme but due to climatic changes, it is reducing continuously. In last century, 75% decline in genetic diversity was witnessed in farmers' fields and it would further decline by about 20% by 2050.

Besides that, genetic resources can be excellent breeding material to develop superior variety in future breeding programmes. They can also be used in different breeding programs in order to increase the genetic base of cultivated crop varieties.

It has been observed that wild relatives have several desirable characteristics like resistance to biotic and abiotic streses, nutritional characteristics, cleistogamy, photo insensitivity and cytoplasmic male sterility (CMS).

In the past few decades, revolutionary approaches and systems have offered a great wealth of genetic and genomics resources that revolutionized research in both model and crop legumes.

A recent study on chickpea presents evidence of severe domestication bottleneck. Efficiency of cultivated population of chickpea is 100 times lesser than that of wild chickpea (Cicer reticulatum and C. echinospermum). In legume crops, study on landraces and wild relatives are significantly benefited by advanced technologies of genomics, phenotyping and computational biology.

The Vavilov Institute of Plant Genetic Resources (VIR), which houses a special genebank in St. Petersburg, Russia, using a mixture of genomics, computational biology and phenotyping to classify the 147 accessions of chickpea from Turkey and Ethiopia. The combination of high-density genotyping data with historical phenotypic information on these VIR landraces allowed chickpea genomes to enter 'agro islands' or 'domestication islands' that display significant associations with multiple phenotypes. These "genomic gems" have also been identified in chickpeas containing co-adapted and co-localized gene complexes. These are LG4 and LG2 in chickpea containing multiple genes/ QTLs related to drought and disease resistance, respectively. WGRS/RADSeq of 90 Cicer accessions, including cultigens, landraces and wild accessions, previously identified a wide collection of 54 genes on LG3 that could have been targeted during modern breeding efforts to manipulate salient characteristics such as flowering time.

Similarly, a genomic segment with an excess of MTAs for agronomically significant traits was observed on LG9 after re-sequencing of 292 accessions in pigeonpea [13].

In a recent study, to understand the genetic relationships between various lentil species/subspecies, a lentil collection comprised of 467 wild and cultivated genotypes originating from 10 different geographical regions was evaluated. A total of 422, 101 high-confidence SNP markers were identified against the reference lentil genome (cv. CDC Redberry). Phylogenetic analysis clustered the germplasm collection into four groups, namely, Lens culinaris/Lens orientalis, Lens lamottei/Lens odemensis, Lens ervoides, and Lens nigricans. Results of this study indicated that L. nigricans is most distantly related to L. culinaris and major differences were observed in six genomic regions with the largest being on Chromosome 1 (c. 1 Mbp) and further additional structural variants are likely to be identified from genome sequencing studies. In order to improve germplasm and for introgression of novel genes, this will provide further insights into the evolutionary relationship between cultivated and wild lentil germplasm.

Guar (Cyamopsis tetragonoloba (L.) Taub.) is primarily grown as an industrial crop due to its high-quality galactomannan gum used as a thickener, flocculant,

emulsion stabiliser and gelling agent. Therefore, the novel set of molecular markers (nSSR) could be adopted as a useful tool to characterize the guar accessions for future breeding programmes.

#### **2.7 Marker assisted backcrossing (MABC)**

Research on legumes has greatly benefited from different available molecular markers in crop plants. Association between molecular markers and plant traits in these crops has introduced a novel approach to breeding that is based on the crossing of selected genotypes and selection of suitable progenies based on associated markers/QTL(s) rather than depending solely on phenotypes. Over the past three decades, the advancement and development of molecular marker technologies have been steady, such as low-throughput restriction fragment length polymorphisms (RFLPs) in the 1980s, high-throughput array-based markers in the 2000s and next-generation ultrahigh-throughput sequence-based marker systems in the 2010s. RFLP, RAPD, AFLP and SSR markers are low-throughput marker systems and are also considered past molecular markers. Besides these, next-generation sequencing (NGS) and genotyping by sequencing (GBS) are high and ultrahigh-throughput marker systems. These are based on low-cost and high-throughput sequencing technologies and are considered as present marker systems.

In cereals, so many outstanding achievements of marker-assisted breeding are available, but in legumes, negligence and lack of genomic resources adversely affected their initial establishment in the field of molecular breeding. Now recent advances in pulse genomics have led to the launch of several marker-assisted breeding projects.

RAPD and RFLP markers were used in five wild lentil taxonomic groups to understand their genetic makeup [47]. A genetic linkage map was also constructed in lentils with RAPD, AFLP and RFLP markers [48].

For shielding the varieties against various biotic and abiotic stresses and for ensuring crop productivity; gene mapping, tagging and marker-assisted selection have vital importance. Identifying and deploying molecular markers/QTLs in a desired background would be a priority. Marker-aided selection (MAS) greatly reduce the time and effort required to recover high levels of resistance from the donor and simultaneously recover the genomes of the recurrent parent. It has become more easier to transfer desirable genes/QTLs from wild relatives to existing cultivars due to MAS and transgenics.

Fusarium wilt (FW) and Ascochyta blight (AB) are two major constraints in chickpea (Cicer arietinum L.) production. The most affordable approach for longterm control of ascochyta blight and fusarium wilt in chickpeas is known to be the use of varieties with high resistance levels.

The availability of molecular markers associated with QTL for ascochyta blight resistance provided an opportunity to introgress the traits into adapted chickpea cultivars. backcrossing between moderately resistant donors (CDC Frontier and CDC 425-14) and the adapted varieties (CDC Xena, CDC Leader and FLIP98-135C) resulted in a variety with improved resistance to ascochyta blight [49].

More recently, five resistant lines representing foc2 gene introgressed into the background of Pusa 256 were reported with the help of foreground selection aided by two SSR markers (TA 37 and TA110). Cultivar Vijay was used as a donor of foc2 gene [50]. Annigeri 1 and JG 74 are elite high-yielding desi cultivars of chickpea, in Karnataka and Madhya Pradesh. in recent years, have become susceptible to race 4 of Fusarium wilt (FW).

A widely grown cowpea variety in Africa was improved by adding drought tolerance, striga and root knot nematode resistance QTLs using SNP markers. The major QTL region on LG 8 was introgressed from cultivar V-16 into the bacterial leaf blight susceptible variety C-152 through marker-assisted backcrossing (MABC) [51]. Similarly, By backcrossing resistance to CpMv gene was transferred into variety C-152. Cowpea mosaic virus (CpMV) was responsible for 80–100% yield loss in cowpea. SSR markers were used for linkage map construction and indicated that two markers MA15 and MA 80 were linked to CpMV resistance.

At ICRISAT in hybrid pigeonpea programmes, markers associated with fertility restoration and CMS are being used. This improved the selection efficiency of hybrid breeding and accelerated the breeding work [52]. In addition, a range of markers, including SSRs and SNPs are now available to enable genetic purity testing of pigeonpea hybrids and their parents. Recently, ICRISAT has launched a collaborative effort with ICAR-IIPR and other NARS institutions/universities to accelerate and target the improvement of ruling mega varieties of pigeonpea in India.

In groundnut breeding, the use of molecular markers in backcross breeding programme accelerated selection of recombinant progenies bearing nematode resistance and high oleic acid. Selection for high oleic acid content in groundnut was facilitated by one CAPS marker along with gel-free SNP assay using HybProbe design for the selection of nematode resistance SCAR, SSR and CAPS marker were used.

Recently in peanuts, two ahfad2 alleles from SunOleic 95R were introgressed into ICGV 05141 using marker-assisted selection. Marker-assisted breeding effectively increased oleic acid and oleic to linoleic acid ratio in recombinant lines up to 44% and 30%, respectively as compared to ICGV 05141. Subsequently, In the marker-assisted backcrossing-introgression lines, a 97% increase in oleic acid, and a 92% reduction in linoleic acid content were observed in comparison to the recurrent parent [53].

As opposed to traditional breeding, gene stacking or pyramiding is a useful strategy for transferring multiple desired genes or QTLs from various parents into a single genotype in the shortest possible time (two to three generations). Molecular markers that may be beneficial for marker-assisted selection and gene pyramiding have been identified through genetic linkage analyses and QTL mapping. The most effective and inexpensive means of combating plant diseases is the use of genetic resistance. Gene pyramiding is thus a sensible approach to creating multiple and enduring resistance. Most successful approach in common bean for wide spectrum control of common mosaic virus is to combine I, bc-u, bc-12, bc-22, and bc-3 genes. SCAR marker was used for MAS. In lentils, molecular marker-assisted gene pyramiding was used for resistance to ascochyta blight and anthracnose. In this research, two genes for resistance to ascochyta blight and the gene for anthracnose resistance in lentil breeding lines were pyramided using linked RAPD marker [49].

#### **2.8 Genome editing**

Crop plant genome editing is a faster-growing technique for inserting specified changes into the genome precisely and with great accuracy. Genome editing has emerged as an alternative approach to conventional plant breeding, and transgenic (GMO) approaches to improve food legumes and their sustainable production. Instead of spontaneous non-specific changes caused by radiation or chemical mutagenesis, crop researchers have long required mutations at specific sites in the genome. This

#### *Recent Advancements in Genetic Improvement of Food Legume Crops DOI: http://dx.doi.org/10.5772/intechopen.106734*

approach allows for site-specific DNA insertion, deletion, modification, or replacement in a living organism's genome. The plant research community has not been widely involved with earlier SSN-specific (sequence-specific) genome-based editing technologies, because of the complex design and labour-intensive assembly of particular DNA binding protein for each target gene. A relatively new and comparatively easier technique for genome editing is CRISPR (clustered regularly interspaced short palindromic repeats) technique which is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defence system. CRISPR genome editing technique is based on Cas9 protein which is an endonuclease. This endonuclease induces double-strand breaks using a guide RNA that is complementary to a target gene [54]. In order to create mutants for inaccessible genes, CRISPR-Cas9 would be a very useful technique. It can mutate multiple loci and make large deletions, thereby speeding the plant breeding without directly adding any transgene. The sequence-specific nucleases-based plant genome editing has a great potential to develop modified crops which can address the increased global food requirements and sustainable agriculture production. CRISPR/ Cas9 was applied first in model legume plants to induce targeted mutagenesis.

A web tool was designed to identify potential CRISPR/Cas9 target sites and also a soybean codon-optimized CRISPR/Cas9 platform to induce mutation at target sites in somatic cells of Glycine max and Medicago truncatula [55]. In a recent study, an efficient CRISPR/Cas9 system was developed for targeted gene mutations in the model legume M. truncatula. A specific sgRNA was designed that targeted medicago phytoene desaturase (MtPDS) gene involved in the carotenoid biosynthesis. Very recently in Cowpea, the representative SNF gene has been effectively disrupted with an efficient CRISPR/Cas9-mediated genome editing. Guide RNAs (gRNAs) for the symbiosis of receptor-like kinase (SYMRK), reached ~67% mutagenesis efficiency in plants with hairy roots, and the formation of nodules in both mutants was totally prevented [56]. Conventional breeding is based on natural genetic variation and rigorous back-crossing systems are needed to incorporate the selected traits into an elite genotype. Unlike conventional breeding techniques, the present diversity does not limit CRISPR because it can directly integrate new mutations. This approach will benefit particularly those crops which have narrow genetic diversity and low variability for traits. Therefore, genome editing can speed up plant breeding programmes by inserting correct and predictable modifications directly in desirable backgrounds. The CRISPR/Cas9 system is especially beneficial because multiple traits can be modified simultaneously.
