**1. Introduction**

Okra (*Abelmoschus esculentus* L. Moench), which belongs to Malvaceae family, is an important fruit vegetable grown throughout the tropics and warmer parts of the temperate zone. It is cultivated in India, Nigeria, Europe, Turkey, Iran, West Africa, Afghanistan, Pakistan, Burma, Japan, Bangladesh, Brazil, China, Ethiopia, Cyprus, United States and all parts of tropics. It has 1.12 million hectare area and 8.7 million tonnes production in the world. It is one the most important traditional vegetable crops of India from production point of view, as India contributes around 73% of total worlds okra production. Okra is one of the important vegetables export from India. India produces annually over 63 lakhs metric tonnes of okra from an area of 5.24 lakh hectares which is valued at Rs. 534,037 lakhs at current market rates [1]. It is fourth most important crop after tomato, brinjal and chilli from seed industry

viewpoints in India. Share of hybrid seed is more than 70% in nearly 6000 metric tonnes seed market in India. Okra is proved to be a very remunerative crop for farmers, but due to Bhendi Yellow Mosaic and Enation Leaf Curl Virus disease its successful production has become a challenge for the farmers all over the country, as most of the previously bred varieties like Parbhani Kranti, P-7, Arka Anamika and Arka have lost the resistance to YVMV and ELCV diseases [2]. Therefore, against viruses development of varieties/hybrids should the continuous process to enhance the crop productivity.

Tender pods of okra are used as delicious vegetable. To a limited extent, it is used in canned, dehydrated and frozen form. It removes constipation when 2-3 fresh pods are eaten regularly. It is often included in weight loss diet as it is both fat and cholesterol free and rich in fibre. It is rich source of protein, calcium, potassium and iodine. Fresh pod contains around 88% water, 0.1% fat, 8% carbohydrate, 1.8% protein and 0.9% fibre. Okra mucilage has potential for use as food, non-food products and medicine. Dried stems and roots of okra are used for cleaning sugarcane juice from which molasses is prepared. The dry seeds are rich source of oil (18-20%) and crude protein (20-23%). Edible oil of okra has pleasant taste and odour and it is high in unsaturated fats such as oleic and linoleic acid. Its ripe, roasted seeds are also used as coffee additive or substitute after grinding. It has a vast potential as one of the foreign exchange earning crops and accounts for about 60% of the export of fresh vegetables excluding potato, onion and garlic. Fresh okra is exported to Middle East, U.K., Western Europe, Singapore and USA. Frozen okra is also exported to U.K. cultivated okra, *A. esculentus* (2n = 130), is natural amphidiploid from chromosome doubling of cross between *A. tuberculatus* (2n = 58) as one parent and *A. ficulneus* (2n = 72) the other probable parent.

Genomic resources are practically absent in *Abelmoschus*, only two mRNA and few coding sequences of this genus are deposited in the public domain [3]. Okra genome is allopolyploid in nature and posses a large number of chromosomes (2n = 56–196) which makes it more complex for genome sequencing. Some, of the complexities in non model genomes like okra can be bypassed by sequencing the transcriptome rather than the genome [4]. mRNA sequencing also known as RNAseq. Has emerged as a powerful tool to identify novel transcript/gene sequences and to develop molecular marker in non-model plants like okra.

#### **2. Begomoviruses infecting okra**

There are at least 27 begomoviruses which infects okra; of which, two viruses i.e. Yellow Vein Mosaic Virus (YVMV) and Okra Enation Leaf Curl Virus (OELCV) most severely affect quality of pod and lowers production by reducing yield [5]. The diseases of begomoviruses are mainly transmitted by insect vector whitefly (*Bemisia tabaci*). Begomoviruses can also be transmitted by grafting; but, seedtransmission or transmission through mechanical inoculation has not yet been established [6].

The yellow vein mosaic disease of okra (YVMD) caused by Bhendi yellow vein mosaic virus (BYVMV) was first reported in 1924 from the erstwhile Bombay Presidency [7]. The begomoviruses native to the New World have only bipartite genomes (having DNA-A and DNA-B components) whereas, of Old World are mostly monopartite (have DNA-A homolog and lacks DNA-B). In India, bhendiinfecting monopartite begomoviruses were mostly associated along with a specific betasatellite, Bhendi yellow vein betasatellite (BYVB). These BYVBs have been reported to be pathogenicity determinant and found responsible for the characteristic yellow vein mosaic symptoms. Minimum16 types of begomoviruses and 4 types

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markers [21].

*Genomic Tools to Accelerate Improvement in Okra (*Abelmoschus esculentus*)*

of beta satellites are found associated with the YVMD in different combinations [8]. Begomoviruses isolated from okra throughout the world are of monopartite nature [9]. However, tomato leaf curl New Delhi virus (ToLCNDV), which is a bipartite begomovirus and bhendi yellow vein Delhi virus (BYVDeV) also a bipartite species

The typical symptom of YVMD is yellowing of veins with in green leaf and if infection becomes severe infected leaves turn completely yellowish. In case of early infection of YVMV there is significant reduction i.e. up to 94 to 100% in terms of yield [11]. Occurrence of infection after 50–65 days of germination reduces the damage and loss by 49–84% [12]. Moreover, the popular varieties of okra in India have become susceptible to YVMV [13], new biotypes of whitefly vectors have surfaced and vectors have become partly resistant to the insecticides [14]. All these factors lead to decrease in overall production of okra in India. Therefore, advance biotechnological and genomic tools like RNA interference (RNAi), genome editing and sequencing along with conventional methods like transfer of resistance from wild sources are required to enhance production of okra under YVMD condition.

Okra Enation Leaf Curl Virus Disease (OELCD) was first identified from Bangalore during the early 1980s in India; OELCD can reduces yield up to 80– 90% in okra [15]. The typical symptoms of OELCD are curling in leaves, thick veins, and reduced leaf surface area. In addition, the disease bearing plants become severely stunted along with small and malformed fruits which make them unsuitable for consumers. This disease is going to be the future menace of okra cultivation and needs a strategic breeding program to evolve resistance against OELCV [8].

**4. Molecular marker development and gene diversity studies in okra**

Isolation of purified DNA is challenging in okra due to the presence of large amounts of mucilaginous acidic polysaccharides like polygalacturonic acid and polyphenols which reduces yield as well as purity of DNA [16]. Presence of impurities in the DNA hinder the downstream processing of DNA like PCR, digestion with restriction enzymes and labelling of DNA segment [17]. Molecular markers has emerged as a potential system for evaluation of genetic variations and associations at inter and intra species level [18, 19]. Most commonly used markers for genetic studies and marker assisted breeding programme are Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism and Simple Sequence Repeat (SSR) [20]. However, okra lacks information on molecular

There are only few studies where markers were used to assess the genetic diversity in okra using general DNA markers [22, 23]. Gene diversity studies reported in okra is listed in **Table 1**. RAPD was initially used in genetic diversity studies between different accessions of okra [23, 26, 27, 32]. Sequence related amplified polymorphism (SRAP) [22] have also been used in okra for diversity analysis studies. SSR markers are an important marker tool in the application of plant genetics and breeding because of their high reproducibility, multiallelic nature, codominant inheritance and good genome coverage [33]. To develop the microsatellite markers in okra, Ravishankar et al. [34] has performed sequencing of genomic DNA employing Roche 454 Titanium pyrosequencing. A total of 979,806 bp data was generated and 61,722 reads were attained. Out of 3735 contigs

*DOI: http://dx.doi.org/10.5772/intechopen.97005*

were characterized from okra [10].

**3. Okra Enation Leaf Curl Virus**

*Genomic Tools to Accelerate Improvement in Okra (*Abelmoschus esculentus*) DOI: http://dx.doi.org/10.5772/intechopen.97005*

of beta satellites are found associated with the YVMD in different combinations [8]. Begomoviruses isolated from okra throughout the world are of monopartite nature [9]. However, tomato leaf curl New Delhi virus (ToLCNDV), which is a bipartite begomovirus and bhendi yellow vein Delhi virus (BYVDeV) also a bipartite species were characterized from okra [10].

The typical symptom of YVMD is yellowing of veins with in green leaf and if infection becomes severe infected leaves turn completely yellowish. In case of early infection of YVMV there is significant reduction i.e. up to 94 to 100% in terms of yield [11]. Occurrence of infection after 50–65 days of germination reduces the damage and loss by 49–84% [12]. Moreover, the popular varieties of okra in India have become susceptible to YVMV [13], new biotypes of whitefly vectors have surfaced and vectors have become partly resistant to the insecticides [14]. All these factors lead to decrease in overall production of okra in India. Therefore, advance biotechnological and genomic tools like RNA interference (RNAi), genome editing and sequencing along with conventional methods like transfer of resistance from wild sources are required to enhance production of okra under YVMD condition.

## **3. Okra Enation Leaf Curl Virus**

*Landraces - Traditional Variety and Natural Breed*

and *A. ficulneus* (2n = 72) the other probable parent.

to develop molecular marker in non-model plants like okra.

**2. Begomoviruses infecting okra**

enhance the crop productivity.

viewpoints in India. Share of hybrid seed is more than 70% in nearly 6000 metric tonnes seed market in India. Okra is proved to be a very remunerative crop for farmers, but due to Bhendi Yellow Mosaic and Enation Leaf Curl Virus disease its successful production has become a challenge for the farmers all over the country, as most of the previously bred varieties like Parbhani Kranti, P-7, Arka Anamika and Arka have lost the resistance to YVMV and ELCV diseases [2]. Therefore, against viruses development of varieties/hybrids should the continuous process to

Tender pods of okra are used as delicious vegetable. To a limited extent, it is used

Genomic resources are practically absent in *Abelmoschus*, only two mRNA and few coding sequences of this genus are deposited in the public domain [3]. Okra genome is allopolyploid in nature and posses a large number of chromosomes (2n = 56–196) which makes it more complex for genome sequencing. Some, of the complexities in non model genomes like okra can be bypassed by sequencing the transcriptome rather than the genome [4]. mRNA sequencing also known as RNAseq. Has emerged as a powerful tool to identify novel transcript/gene sequences and

There are at least 27 begomoviruses which infects okra; of which, two viruses i.e. Yellow Vein Mosaic Virus (YVMV) and Okra Enation Leaf Curl Virus (OELCV) most severely affect quality of pod and lowers production by reducing yield [5]. The diseases of begomoviruses are mainly transmitted by insect vector whitefly (*Bemisia tabaci*). Begomoviruses can also be transmitted by grafting; but, seedtransmission or transmission through mechanical inoculation has not yet been

The yellow vein mosaic disease of okra (YVMD) caused by Bhendi yellow vein mosaic virus (BYVMV) was first reported in 1924 from the erstwhile Bombay Presidency [7]. The begomoviruses native to the New World have only bipartite genomes (having DNA-A and DNA-B components) whereas, of Old World are mostly monopartite (have DNA-A homolog and lacks DNA-B). In India, bhendiinfecting monopartite begomoviruses were mostly associated along with a specific betasatellite, Bhendi yellow vein betasatellite (BYVB). These BYVBs have been reported to be pathogenicity determinant and found responsible for the characteristic yellow vein mosaic symptoms. Minimum16 types of begomoviruses and 4 types

in canned, dehydrated and frozen form. It removes constipation when 2-3 fresh pods are eaten regularly. It is often included in weight loss diet as it is both fat and cholesterol free and rich in fibre. It is rich source of protein, calcium, potassium and iodine. Fresh pod contains around 88% water, 0.1% fat, 8% carbohydrate, 1.8% protein and 0.9% fibre. Okra mucilage has potential for use as food, non-food products and medicine. Dried stems and roots of okra are used for cleaning sugarcane juice from which molasses is prepared. The dry seeds are rich source of oil (18-20%) and crude protein (20-23%). Edible oil of okra has pleasant taste and odour and it is high in unsaturated fats such as oleic and linoleic acid. Its ripe, roasted seeds are also used as coffee additive or substitute after grinding. It has a vast potential as one of the foreign exchange earning crops and accounts for about 60% of the export of fresh vegetables excluding potato, onion and garlic. Fresh okra is exported to Middle East, U.K., Western Europe, Singapore and USA. Frozen okra is also exported to U.K. cultivated okra, *A. esculentus* (2n = 130), is natural amphidiploid from chromosome doubling of cross between *A. tuberculatus* (2n = 58) as one parent

**86**

established [6].

Okra Enation Leaf Curl Virus Disease (OELCD) was first identified from Bangalore during the early 1980s in India; OELCD can reduces yield up to 80– 90% in okra [15]. The typical symptoms of OELCD are curling in leaves, thick veins, and reduced leaf surface area. In addition, the disease bearing plants become severely stunted along with small and malformed fruits which make them unsuitable for consumers. This disease is going to be the future menace of okra cultivation and needs a strategic breeding program to evolve resistance against OELCV [8].

#### **4. Molecular marker development and gene diversity studies in okra**

Isolation of purified DNA is challenging in okra due to the presence of large amounts of mucilaginous acidic polysaccharides like polygalacturonic acid and polyphenols which reduces yield as well as purity of DNA [16]. Presence of impurities in the DNA hinder the downstream processing of DNA like PCR, digestion with restriction enzymes and labelling of DNA segment [17]. Molecular markers has emerged as a potential system for evaluation of genetic variations and associations at inter and intra species level [18, 19]. Most commonly used markers for genetic studies and marker assisted breeding programme are Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism and Simple Sequence Repeat (SSR) [20]. However, okra lacks information on molecular markers [21].

There are only few studies where markers were used to assess the genetic diversity in okra using general DNA markers [22, 23]. Gene diversity studies reported in okra is listed in **Table 1**. RAPD was initially used in genetic diversity studies between different accessions of okra [23, 26, 27, 32]. Sequence related amplified polymorphism (SRAP) [22] have also been used in okra for diversity analysis studies. SSR markers are an important marker tool in the application of plant genetics and breeding because of their high reproducibility, multiallelic nature, codominant inheritance and good genome coverage [33]. To develop the microsatellite markers in okra, Ravishankar et al. [34] has performed sequencing of genomic DNA employing Roche 454 Titanium pyrosequencing. A total of 979,806 bp data was generated and 61,722 reads were attained. Out of 3735 contigs


#### **Table 1.**

*Gene diversity studies in okra.*

obtained from assembled reads, a total of 2708 contigs had microsatellites. Finally 402 microsatellites were used for selection of 50 SSR primers for amplification in okra. This is the first report on the development of genomic SSR markers in okra using next-generation sequencing technology.
