**3. Identification of mutagenic site through molecular marker**

Markers have been used for cultivar recognition as the first light of forward genetics. Markers fall into three major categories as visually measurement of traits, gene product as well as DNA test known as morphological markers, biochemical markers as well as molecular markers respectively [75]. Classical breeding can be fast-tracked using molecular marker approaches for identification of mutagenic regions and to access the variations inside genome [76]. These markers lies inside the genetic region or nearby it. Newest genotyping approaches like genotyping by sequencing (GBS) made it easier to identify even a single base pair change as single nucleotide polymorphism (SNP).

#### **3.1 Morphological markers**

Most of the induced mutants have been released as cultivars by selection through morphological markers. These markers based upon agronomic traits such as maturity, height, early flowering, fruit appearance, seed quality as well as resistance to diseases that can be monitored easily as a result of their epiphytotic character. Huge morphological, physiological as well as ecological differences has been existed in cultivated rice genomes. It is a general approach employed to determine genotypic relationship [77].

Detection of morphological traits is performed by statistically method. Multivariate methods have statistical approaches that widely used in telling the intrinsic variation among various crop genotypes. Multivariate analysis has been reported for study of genetic diversity in numerous crops such as barley [78], sorghum [79], wheat [80], peanut [81] and rice [82].

#### **3.2 Genetic markers**

Mutant phenotypes were usually recognized depend upon their morphological characteristic. But morphological markers are not steady due to less heritability

**15**

*Genetic Variability through Induced Mutation DOI: http://dx.doi.org/10.5772/intechopen.95027*

breeding as well as genetics [86].

*3.2.1.1 SSRs as a sequence based marker*

*3.2.1 Types of genetic marker*

along with complex genetic nature [83]. Then novel method have been developed depend upon genetic information of DNA. This procedure is a faster and more reliable as compared to other methods. Molecular markers depend upon genetic variant in the genome [84]. DNA-based markers have basically replaced previous biochemical markers as importance of DNA analysis has been reported in plants [85]. DNA markers are largely scattered across the entire genome due to larger in quantity [75]. Genetic markers perform main role for reorganization of heritable traits in plant

Numerous types of molecular markers have been utilized. Molecular markers include restriction fragment length polymorphism (RFLP) [87], random amplified polymorphic DNA (RAPD) [88], amplified fragment length polymorphism (AFLP) [89], inter-simple sequence repeat (ISSRs) [90], microsatellite or simple sequence repeats (SSRs) [91] and single nucleotide polymorphisms (SNPs) [92] are currently

These markers are group of tedious DNA chain typically two to six base pairs. It is a form of VNTRs (Variable Number Tandem Repeats) [93]. These markers are wellknown as STRs (short tandem repeats). The rate of different STRs length is feature of microsatellite loci in rice [75]. They consist of dinucleotide; trinucleotide as well as tetranucleotide repeats for genetic analysis. Dinucleotides are the key form present in most vertebrates. Trinucleotide repeat are rich in plants [94]. Microsatellite markers are believed to be suitable over different array of markers due to following reasons. These are scattered all over the genome of extremely conserved region. These markers have various qualities of simplicity, high polymorphism, rapidity as well as stability. These markers have been model for examination of germplasm, genetic diversity [95], heterosis, purity test, gene mapping, fingerprints assembly, phylogenetic comparison as well as marker aided selection [75, 95]. A random collection of SSRs assist in estimation of rice genetic diversity and rice cultivar classification without mistakes [96]. Particularly SSRs markers have been extensively employed in rice genetic analysis for high allelic detection [97]. Microsatellite exposed unreliable level of genetic relation-

SNPs (single nucleotide polymorphisms) signify a strong group of genetic markers [99] among different categories of molecular markers due to following reason [100]. These markers detect single-base pair location depend upon sequence variation in genomes [101]. SNPs markers offer a huge marker density in genomes [102]. SNPs markers have achieved significant importance in plant genetic analysis due to their brilliant genetic qualities, genetic diversity, evolutionary interaction [103], high throughput genetic mapping [104], population substructure [105], genomewide linkage disequilibrium [106] as well as association mapping [107]. Availability of high quality reference genome sequence made it easier to scan out mutation by re-sequencing the species genotypes through next generation sequencing (NGS) approaches and to identify the variation in targeted genotype through mapping

Classically, a quite large sequencing attempt is faithful to recognize polymorphic location in a genome among a set of various breeding lines [109]. A precise

accessible to evaluate the diversity and variability at the DNA level.

ship among the domesticated as well as wild collection of rice [98].

*3.2.1.2 SNPs as genetic markers from high-throughput sequencing*

techniques as genome-wide association mapping [108].

#### *Genetic Variability through Induced Mutation DOI: http://dx.doi.org/10.5772/intechopen.95027*

along with complex genetic nature [83]. Then novel method have been developed depend upon genetic information of DNA. This procedure is a faster and more reliable as compared to other methods. Molecular markers depend upon genetic variant in the genome [84]. DNA-based markers have basically replaced previous biochemical markers as importance of DNA analysis has been reported in plants [85]. DNA markers are largely scattered across the entire genome due to larger in quantity [75]. Genetic markers perform main role for reorganization of heritable traits in plant breeding as well as genetics [86].

### *3.2.1 Types of genetic marker*

*Genetic Variation*

*63 immunity associated genes*

*VvMLO3; VvMLO4*

*GhCLA and GhPEBP*

**Table 1.**

**Gene targeted**

**3. Identification of mutagenic site through molecular marker**

*Induced mutation in plants using targeted genome editing method CRISPR/Cas9.*

**Vector: promoter Transformation** 

P201N-Cas9: U6 promoter

pYLCRISPR/ Cas9Pubi-H: U6 promoter

gRNA-Clpk1: AtU6 promoter

pRGEB32-GhU6.7: cotton U6 promoter

*HvITPK1* pYLsgRNA-OsU6

*Clpsk1* pRGEBB320cas9-

pYLCRIPSR/Cas9-N vector: *AtU3b* and *AtU6–1* promoter

**method: promoter**

Agrobacterium ID1249 strain

Agrobacterium strain GV3101

Agrobacterium strain AGL1

Agrobacterium strain EHA105

Agrobacterium strain GV3101

**Plant variety Mutation** 

Tomato RG-PtoR or RG-prf3

Grape wine PN40024 (*Vitis vinifera*)

Barley model cultivar Golden Promise

Watermelon Sumi 1

Cotton Jin668 (*Gossypium hirsutum*)

**nature**

Short Indels

Short Indels

Insertion mutation

Point mutation

Lnockout [73]

**Reference**

[70]

[71]

[72]

[74]

nucleotide polymorphism (SNP).

sorghum [79], wheat [80], peanut [81] and rice [82].

**3.1 Morphological markers**

**3.2 Genetic markers**

Markers have been used for cultivar recognition as the first light of forward genetics. Markers fall into three major categories as visually measurement of traits, gene product as well as DNA test known as morphological markers, biochemical markers as well as molecular markers respectively [75]. Classical breeding can be fast-tracked using molecular marker approaches for identification of mutagenic regions and to access the variations inside genome [76]. These markers lies inside the genetic region or nearby it. Newest genotyping approaches like genotyping by sequencing (GBS) made it easier to identify even a single base pair change as single

Most of the induced mutants have been released as cultivars by selection through morphological markers. These markers based upon agronomic traits such as maturity, height, early flowering, fruit appearance, seed quality as well as resistance to diseases that can be monitored easily as a result of their epiphytotic character. Huge morphological, physiological as well as ecological differences has been existed in cultivated rice genomes. It is a general approach employed to determine genotypic relationship [77]. Detection of morphological traits is performed by statistically method. Multivariate methods have statistical approaches that widely used in telling the intrinsic variation among various crop genotypes. Multivariate analysis has been reported for study of genetic diversity in numerous crops such as barley [78],

Mutant phenotypes were usually recognized depend upon their morphological characteristic. But morphological markers are not steady due to less heritability

**14**

Numerous types of molecular markers have been utilized. Molecular markers include restriction fragment length polymorphism (RFLP) [87], random amplified polymorphic DNA (RAPD) [88], amplified fragment length polymorphism (AFLP) [89], inter-simple sequence repeat (ISSRs) [90], microsatellite or simple sequence repeats (SSRs) [91] and single nucleotide polymorphisms (SNPs) [92] are currently accessible to evaluate the diversity and variability at the DNA level.
