Bioinformatics – Technology Developed

Tamil Nadu Agricultural University > Centre for Plant Molecular Biology and Biotechnology > Bioinformatics – Technology Developed

RESEARCH

  1. Marker – assisted Breeding
  2. Bioinformatics

1. MARKER-ASSISTED BREEDING

1.1 Abiotic Stress

Promising drought tolerant rice cultures viz., CPMB ACM 04 004, CPMB ACM 04 006 and PM 01 011, PM 03 002 and RM 04 001 were developed through marker aided selection (MAS) and farmers participatory plant breeding approach in the Department of Plant Molecular Biology and Biotechnology, CPMB with the help of Department of Plant Breeding and Genetics, AC&RI, Madurai, ARS, Paramakudi and CSRC, Ramnad. Among these drought tolerant rice lines, PM 01 011 performed better over years and across locations. It recorded an average grain yield of 3891 kg/ha, which is 14.40 per cent increased yield over the ruling check variety PMK (R) 3 (3401 kg/ha) and is accepted for release as location specific variety for Ramnad and Sivagangai districts during the year 2008. This culture was released as PMK(R) 4 or Anna(R) 4 in 2009.

Highlights of PMK (R) 4 (PM 01 011):

  • Higher productivity of 3 .6 t/ha.
  • Drought tolerant.
  • Semi dwarf, erect, non-lodging.
  • Short duration (100 – 105 days) which is a week earlier than PMK ( R )
  • Suitable for September – October sowing (North East Monsoon) in rainfed rice areas of Ramanathapuram and Sivagangai districts.
  • Long slender white rice with high head rice recovery (62.1 %)

1.2 Biotic Stress 

  1. Bacterial Blight resistance in rice:   

Evolving high yielding rice genotypes with durable resistance to bacterial blight (BB) is pertinent considering the extensive damage caused by the disease in most of the rice growing regions. Two high yielding BB susceptible indica rice cultivars, ADT43 and ASD16 popular among farmers and consumers across South India have been introgressed with three BB resistance genes xa5, xa13 and Xa21 from isoline IRBB60 using functional markers.

  1. Culture on Farm Trials:
  1. AD (Bio) 09518 (ADT 43 x IRBB 60-5-1) – Bacterial Blight resistant rice culture

A short duration (115-120 days) rice culture, AD (Bio) 09518 (ADT 43 x IRBB60-5-1) with the yield potential of 5767 kg/ha and high resistance to bacterial blight has been developed through marker-assisted pedigree breeding. It has medium slender grains with high head rice recovery (80.6%). The culture, AD (Bio) 09518 has a amylose content of 25.48 per cent.

On cooking, the rice quality is equivalent to CO 51. The culture has soft gel and good volume expansion. This culture was tested under adaptive research trial (ART-Rice 14/2015-16, 2016-17, Special transplanted Early (May-June sowing) for the two consecutive years. In the first year ART 14 (2015-16), the results from 55 locations across 15 districts revealed that AD (Bio) 09518 recorded an average grain yield of 5999 kg/ha. In the second year ART 14 (2016-17), AD (Bio) 09518 had given an average yield of 5439 kg/ ha in 113 days from 26 locations and manifested on par yield with CO 51 (5484 kg/ha) and ADT 43 (5426 kg/ha). Under artificial screening in green house condition both at TNAU, Coimbatore and Indian Institute of Rice Research, Hyderabad, it showed high resistance to bacterial blight. Out of the 12 pathogenic races tested, the culture is resistant to almost all (11) races of the BB pathogen. The culture also showed moderate resistance to leaf folder.

  1. Biotic stress tolerance in rice:

The aim is to pyramid  genes for Gall Midge (Gm1, Gm4), Blast (Pi54 , Pi9) and  bacterial blight (xa13, Xa21, Xa33, Xa38) resistance in rice varieties CO43  and ASD16 . The recipient lines were susceptible to the above stresses.  The donor lines viz., B95-1 X Tetep (Xa21 and Pi54), B95-1 X Kavya (Xa21 X Gm1), B95-1 X Abhaya (Xa13 and Gm4) and VRP1 (Pi9) were utilized for introgression studies. Molecular markers tightly linked to the resistance genes were utilized in the present study. Pyramiding six genes (Gm1+Gm4+xa13+Xa21+Pi9+Pi54) has been completed. The improved genetic stocks with multiple genes are currently used in crop improvement programmes.

  1. Marker assisted breeding for Sorghum shoot fly resistance

Marker assisted back cross breeding programme was initiated to incorporate the QTLs for shoot fly resistance in CO(S)28 and K8. During the year 2016, the crossing programme between the donor line (IS18551) and recipient lines (CO(S) 28 and K8) was completed. True F1 hybrids were identified using molecular markers and the selected F1 plants were crossed to the recurrent parent (CO(S) 28) for getting BC1F1 seeds.During the year 2017, the selected BC1F1 plants were crossed to the recurrent parent (CO(S) 28) to get BC2F1 seeds. In case of variety K8, the selected F1 plants were crossed to the recurrent parent (K8) to get BC1F1 seeds.During Kharif 2018, BC3F1 of the cross IS 18551X CO (S) 28 was completed and selfed. In the case of variety K8, the second back cross is in progress. The seeds were harvested during the November 2018. BC3F2 generation involving the recurrent parent C0(S)28 and BC2F1 generation with K8 as recurrent parent was studied in summer 2019. Subsequently, BC3F3 generation involving the recurrent parent C0(S)28 and BC2F2 generation with K8 as recurrent parent is currently being evaluated in Rabi 2019.

  1. Mapping of shoot fly resistance QTLs in sorghum

During Rabi 2016, crossing programme was initiated between the shoot fly resistant line IS2205 and K8. F1 generation was raised in summer 2017. Subsequently F2 generation was studied in Kharif 2017. A total 120 F3 lines are evaluated for shoot fly resistance and agronomic traits in this season (Summer 2018). A total of 186 SSR markers were tested for   polymorphism between IS2205 and K8. Out of186 markers, 102 SSR markers showed polymorphism. Polymorphism is about 55%.

Phenotypic and genotypic data for mapping population of 120 F2:3 families were subjected to QTL analysis using QTL IciMapping sofware and also the maps were obtained. Total of 34 QTLs viz., twelve for oviposition (no. of eggs/ seedling on 14 and 21 DAE), five for dead heart, four for abaxial trichome density on leaves, three for adaxial trichome density on leaves, two for leaf glossiness and eight for seedling vigour were detected by Composite interval mapping. The logarithm of the odds (LOD) and phenotypic variation (R2 %) values of QTLs ranged from 2.53 to 14.20 and 3.87 to 29.56 per cent, respectively.

Plant height variations – variety K8 (1.5m), IS 18551 (2.2m), F1 (3 m)

Stem thickness – K8, IS 18551, F1 of K8 X IS 18551

  1. Enrichment of nutritional quality in Maize through Molecular Breeding

Maize is an excellent nutritional source and is consumed as a staple food in different parts of the world, including India. Developing a maize genotype with a combination of higher lysine and tryptophan, along with β-carotene, can help alleviate the problem of protein-energy malnutrition (PEM) and vitamin A deficiency (VAD). Marker-assisted backcross breeding is attempted for introgression of β carotene trait from CE 477 into UMI 1200 and UMI 1230 (Superior inbreds of CO6 maize hybrid) inbred lines. The marker assisted breeding programme resulted in the development of Maize hybrid ACM M15009 with the parentage of UMI1200β+ x UMI1230β+ which is nominated for the Multi-location testing in Tamil Nadu. The hybrid having higher yield of 10730 kg/ha which is 11.3 % increase yield over the existing hybrid CO6 with the β carotene content of 9.60 µg/g

Pyramiding of QPM and high β carotene is take up by crossing the heterozygotes progenies of BC3F1 of UMI1200×VQL 1 & UMI1200×CE 477 and UMI1230×VQL 1 & UMI1230×CE 477 lines. Heterozygote intercross progenies are selected by using crtRB1 gene specific maker and σ2 locus specific umc1066 marker. The selected progenies are selfed to select double homozygotes pyramided lines having beta carotene and QPM traits. The stabilized progenies having QPM and high β carotene in the UMI 1200 and UMI 1230 background are being tested for hybrid development.

  1. DNA fingerprinting and barcoding of varieties and hybrids and pre-release cultures for varieties/hybrids identification and notification

DNA fingerprinting of varieties and hybrids are mandatory for notifications of a variety/hybrid by Central Sub- Committee and Protection of Plant Varieties and Farmers’ right (PPV&FRA). The DNA fingerprinting protocols for the newly released verities/hybrids in various crops are attempted using publically available SSR markers. The conserved sequences like rbcL, MatK and ITS  are used for barcoding of Horticultural Crops since some of the crops not having enough informative SSR markers. The marker information will be cataloged in the form of a database for future references.

  1. Genome-wide identification of rice long noncoding RNAs responsive to Xanthomonas oryzae infection
  • BB resistant (Improved Samba Mahsuri (RP Bio-226)) and susceptible (ADT38) rice varieties were grown at standard field conditions and the leaf sample collected was given for RNA isolation and sequencing after giving Xanthomonas oryzae infection at the time point of 6 hr.
  • Six transcriptome datasets which includes accession id GSE108504, GSE75131, GSE67958, GSE95668, GSE58385 and GSE57670 available in NCBI GEO database for the rice infected with bacterial blight have been retrieved.
  • Approximately 1200 long non-coding RNA were found on differential expression analysis of lncRNA in susceptible and resistant varieties

8. Whole genome sequencing of blackgram (CO5) using next‐generation sequencing technologies

The Variety, CO5 have been chosen for whole genome sequencing and development of genomic resources of blackgram. Retrieved a total of 196 SSR motifs and their primer sequences, which was used for constructing linkage map of adzuki bean, from NIAS Genebank. Followed by, we have developed an initial in‐house database with the retrieved SSR markers of the adzuki bean. The database is developed with three different search options viz., types of repeat (di, tri, tetra, penta, hexa and composite).Types of markers (simple or  olymorphic) and search by primers or motif. The users could retrieve the information on (i) types of motifs, (ii) forward and reverse primers, (iii) chromosomal location, etc. This database was initiated to develop using MySQL as backend tool. PHP and CSS were using for developing user interface.

2. Bioinformatics

 2.1 Database Development 

  1. TNAU Rice

A database comprising of the rice varieties released from Tamil Nadu Agricultural University (TNAU),Coimbatore, India. Backed by MS-SQL, and ASP-Net at the front end, this database provide information on both quantitative and qualitative descriptors of the rice varities inclusive of their parental details. Enabled by an user friendly search utility, the database can be effectively searched by the varietal descriptors, and the entire contents are navigable as well. The database comes handy to the plant breeders involved in the varietal improvement programs to decide on the choice of parental lines. TNAURice is available for public access at (http://www.btistnau.org/germdefault.aspx).

  1. Establishment of MilletDB:

    Millets are small seeded annual grasses and grow well, under the marginal conditions of soil fertility and moisture, which provide staple food for millions of people. Breeding research in millets, habitually engages different species, cultivars, and varieties. This makes an immense challenge to maintain the different varieties and their identification. On account of this fact, the MilletDB clearly brings out the variations of different morphological traits of the TNAU released varieties. These database covers information on various qualitative and quantitative traits on maize, sorghum, cumbu, panivaragu, samai, tenai, kuthiraivali, ragi and varagu. MilletDB is an Internet-accessible database, which is designed by using the front-end language PHP and back-end as MySQL and is equipped with extensive search options. It is a user-friendly database and made publicly available, to improve the research and development of millet crops by making the wealth of available millet varieties.

  1. Establishment of oil-seeds Database:

The  oilseed  database  brings  out  the  variations in different morphological traits of the TNAU released varieties. The   databases  cover  information  on  various   qualitative   and  quantitative   traits  on  oilseeds ( Groundnut, sesame  and sunflower ) varieties. Oilseed database is an Internet-accessible database, which is designed by using the front-end language PHP and back-end as MySQL and is equipped with extensive search options. These are user-friendly databases and are publicly available   http://www.btistnau.in).

 

Tools and Databases Developed URL
TNAU Rice http://btistnau.in/Search.php?category=RICE
TNAU Oilseeds http://btistnau.in/oilseeds/index.html
TNAU Pulses http://btistnau.in/Pulses/index.html
TNAU Millets http://btistnau.in/MilletCrop.php
Anti-inflammatory plants Database http://btistnau.in/Anti-inflammatory/index.php
RLncRNABase http://btistnau.in/RLncRNABase/index.html
813 entries of EST/ Full length cDNA sequences information generated and submitted to NCBI by scientists of TNAU https://www.ncbi.nlm.nih.gov/nuccore/?term=TNAU

 2.2 Tools Development 

  1. Comparative codon usage analysis Tool:

A tool for comparative codon usage analysis has been developed. The compares the frequencies of codon usage that code for the same amino acid (synonymous codons), that can be used to assess whether a sequence shows a preference for particular synonymous codons against the selective genome. ( http://btistnau.in/Codon/front.html) 

  1. Development of a sequence based non-coding RNA prediction tool:    

Non-coding RNAs (ncRNAs) are functional RNA molecules that are transcribed from DNA but are not translated into proteins. In general ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. There are about 2700 noncoding RNA families that are classified for rice in RFAM database. Major ones being, 5s rRNA, transfer RNA, plant signaling etc. Sequence based query tool have been developed which implements a blast like algorithms to search and display the database sequences having identical or similar sequences with other details such their RNA family ID, accession number description, sequence range etc. This tool includes information about 5 major families and developed using PHP.

  1. Genome wide SSR Polymorphism analysis

The databases on SSR markers in rice, wheat and adzuki bean are being developed. Markers data was mined from published literatures and online databases. A total of 196 SSR motifs and their primer sequences, which was used for constructing linkage map of adzuki bean, were retrieved from NIAS Genebank. Followed by, we have developed an initial in‐house database (named VigSSR) with the retrieved SSR markers of the adzuki bean. The database is developed with three different search options viz., detailed search, quick search and reference search. The users could retrieve the information on (i) types of motifs (ii) forward and reverse primers (iii) annealing temperature and (iv) expected product size. VigSSR was developed using MySQL as backend tool. PHP and CSS were used for developing user interface. Developing database on polymorphic SSR markers in rice and wheat is in progress.

2.3 Next Generation Genome Sequencing

  • Whole genome sequencing and transcriptome profiling of millets and pulses have been initiated.
  • Identifying microsatellite markers for important traits against biotic and abiotic tolerance.
  • Genome wide comparison of major food crops for identifying novel genes that are responsible for tolerance against biotic and abiotic stress.

2.4 Structural Bioinformatics

  1. Strategies to explore host-pathogen interaction in cotton:

Cotton is a major field crop in many countries, constituting a valuable cash crop for many small holders in developing countries. It is an important industrial crop in both developed and developing countries. Cotton is known to suffer from number of diseases caused  by fungal, bacterial and viral origins. There is now more relative importance for different diseases may be air borne foliar diseases like grey mildew, alternaria leaf spot, myrothecium leaf spot, bacterial blight, rust, cotton leaf curl virus.

 Recent breakthrough in structural and bioinformatics strategies have enabled greater integrated understanding of complex plant-pathogen interactions. The study on modelling and docking of pathogenic protein and host proteins with and anti-microbial peptides /new lead molecules have shown to possess antimicrobial activity. As results, the promising lead molecules may serve as one of the potential fungicide for cotton fungal disease.

  1. Bioinformatics studies of Protein – Ligand interactions in staple crops:

      In order to keep pace with increasing human population growth, rice yields need to be doubled by 2050. A major obstacle in achieving optimal yields is the occurrence of rice diseases caused by numerous fungal, bacterial, and viral pathogens. For example, Rice blast, sheath blight, and bacterial blight are the three most devastating diseases in Oryza sativa.  Rice blast epidemics often result in between 10% and 30% yield loss, which on a lower estimate, would be enough rice to feed 60 million people for 1 year.  Bioinformatics studies on the devastating plant diseases helps to understand the mechanism of action in effect of cost and time.

    Computational strategies will be developed to understand the specificity and selectivity of proteins and their interactions in the major crop diseases. Efforts will be laid to design inhibitors to resist plants from various pathogens.   Further, comparative genomic studies will be performed to assign protein function and structure for putative proteins which may have role in plant diseases.