DEVELOPMENT AND VALIDATION OF EST DERTVED SSR MARKERS WITH FUNCTIONAL RELEVENCE TO BIOTIC AND ABIOTIC STRESS RESPONSES IN GROUNDNUT 1982
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Date
2015-03
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JAU, JUNAGADH
Abstract
Availability of polymorphic markers in a crop species is a prerequisite for
genetic improvement through marker-assisted selection, construction of high density
genetic linkage map and mapping of Quantitative Trait Loci (QTL). Simple sequence
repeat (SSR) markers are widely employed in molecular breeding programmes and
thus are needed to be developed and validated. Conventional methods for developing
SSR markers are laborious and expensive. The alternative and cost effective way is to
explore the existing public databases harboring abundant number of genomic and
genie (expressed sequence tag, EST) sequences for the development of new SSR
markers. The SSR markers developed from ESTs leverages functional aspect of
transcripts and thus associated with its function. Insufficient number of polymorphic
molecular markers is an obstacle for current molecular breeding research in groundnut
(Arachis hypogaea L.), an important legume grain as well as oilseed cash crop of the
semi-arid tropics. It becomes exceedingly essential to develop more number of
polymorphic SSR markers for potential use in groundnut improvement. Therefore, the present study was carried out to develop EST-SSRs from publically available
groundnut EST resources.
In the present study, a total of 178490 EST sequences of Arachis hypogaea ^ere downloaded from NCBI public databases and pre-processed for assembling. In
order to find novel non-redundant EST-SSR markers, 7170 publically available SSR
primer sequences were subjected to sequence similarity search with the downloaded EST sequences. A total of 23696 (13.28%) very similar sequences were excluded and
the remaining non-redundant sequences (154794) were further pre-processed for removai of low corapiexity sequences, poiy A and poly T tail and vector sequences. A total of 138628 high quality sequences were assembled using TGICL software, which yielded 16424 unigenes comprising of 13428 (81.76%) contigs and 2995 (18.24%) singletons with an average length of 857 bp and N50 value of 942 bases. The assembly by TGICL software reduced the redundancy by 82.21% A total of 2784 sequences were indentifled from unigenes which harbored 3373 SSR motifs The number of sequences harboring more than one SSR was 487 (17.49%) and 289 (10.38%) sequences comprised of compound SSRs. The avemge frequency of SSR was found to be one in 4.17 kb which was relatively higher frequency than previous
reports in groundnut. Regarding functional annotation, 2784 unigenes harboring SSR motifs were subjected to BIast2G0, an online tool for functional annotation. Among the 2784 un,genes, 2027 (72.81%) unigenes were annotated and assinned f . ontology (GO) temrs (4124) were ascribed to unigenes and categorizeriUrr
components (391), molecular functions (1120) and hiol • , "^^I'tilar ntaximum homology of groundnut unigenes through aiyclne n,ax (48.2%) followed by Cto orterimm, (16.6o/„). and the sequences of rep^ ml^C rl'XT'''' motifs were categorized in to class I flonap tu ^ (^^-47%) SSR motifs in to short length range class II a' ^ <«»"%) SSR were the most predominant with 33.86% foUowel 77 "-ofifs Tetra-, penta- and hexa-nucleotide repeats meas L (27-51%). r ~AAT/ATT (4.80%) and 1^1" ^^<='ATG (fss^ assembly. The searrvi, ^ repeats wert^ i
~ -* '■ i. .N. ..z,Pnmers were designed hv BatchPrimer3 tool and 2456 • 2784 SSR com • • Pnmers were designed A ^'"^"8 sequences by 5>ica. A set of . 366 pr,mer pairs with
functional relevance to biotic and abiotic stresses were selected, synthesized and
screened for polymorphism in eleven genotypes which represents the parents of six
mapping populations. A set of 339 (92.62%) primer pairs yielded scorable amplicons
and 39 (10.66%) primer pairs showed polymorphism among eleven parental
genotypes. The 339 primers detected 2 to 12 alleles with an average of 3.77 alleles per
marker where as among the 39 polymorphic primers yielded 2 to 12 alleles with an
average of 5.10 alleles per marker. The Polymorphic Information Content (PIC) value
for these 39 polymorphic markers ranged form 0.028 to 0.375 with an average of
0.325 per marker. The polymorphism screening indicated that compound SSRs were
the most predominantly polymorphic (26.09%) followed by di-nucleotide (13.34%)
and tri-nucleotide (11.72%) motifs. Among the SSR motif repeats, di-nucleotide
repeat SSR markers showed higher PIC value (average 0.357 per marker) followed by
penta (average 0.335 per marker) and compound (average 0.332 per marker).
The newly designed novel SSR markers have added to the repository of
groundnut genomic resources utilizing almost all the available ESTs in public
databases as on available till date. The EST-SSR markers showing polymorphism in
the parental genotypes could be further screened for polymorphism on a panel of
mapping population segregating for biotic or abiotic stress for finding markers linked
with that trait. These EST-SSRs could also be effectively utilized for saturation of
genetic map or transcript map, QTL mapping and marker-assisted selection for
cultivated groundnut.
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BIOTECHNOLOGY