IN SILICO MINING OF SSRs FOR MAPPING OF GYNOECY IN BITTER GOURD (Momordica charantia L.)
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Date
2021-04
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University of Horticultural Sciences, Bagalkot. (College of Horticulture, Bagalkot).
Abstract
Bitter gourd is nutritional and medicinally adorable vegetable. Gynoecy is a
condition where flowering node bears only pistillate flowers. Use of gynoecious line
as a female parent would provide higher yield and high genetic purity. However, these
gynoecious lines are influenced by environment and are governed by recessive genes
because of which transferring into elite background is a difficult. So, identification of
markers linked gynoecy is of more importance. An investigation on in silico mining
of Simple Sequence Repeats (SSRs) from the publicly available restriction associated
DNA (RAD) sequence and transcriptome (RNA-seq) of bitter gourd was carried out
for mapping of gynoecy in bitter gourd (Momordica charantia L.). RAD sequence
assembly of 418832 contigs with mean contig size of 684 bp and N50 contig length of
2383 bp was generated from the quality filtered 19,495,170 paired end RAD-seq reads
of gynoecious like K-44 (ERR2214863). Similarly, filtered RNAseq reads of
gynoecious (Gy323; SRR947759 and SRR953077) and monoecious line (DRAR-1;
SRR950973 and SRR953078) were assembled into 56070 and 51007 unigenes
respectively. Assembled unigenes of gynoecious bitter gourd exhibited median contig
length of 468 bp, average contig size of 867.55 bp, GC % of 41.19 and N50 contig
length of 1553 bp. Monoecious bitter gourd unigenes exhibited median contig length
of 440 bp, average contig size of 917 bp, GC% of 41.33 and N50 contig length of
1801 bp. In silico polymorphic SSRs were mined from the assembled RAD-seq
genome and transcriptome using CandiSSR bioinformatics pipeline. A total of 3937
gSSRs comprising di, tri, tetra, penta and hexa nucleotide motifs were identified from
1575 RAD-seq assembled contigs. Among them 1591 were polymorphic. A total of
398 eSSRs consisting of di, tri, tetra, penta and hexa nucleotide motifs were predicted
across both gynoecious and monoecious bitter gourd lines from assembled
transcriptome of bitter gourd gynoecious line (Gy323) and monoecious line (DRAR1). Among them, 222 SSRs were polymorphic. Highest polymorphic SSRs were
found on chromosome 08 (36), followed by chromosome 09 (29). Least polymorphic
SSRs were identified on chromosome 01 (1), followed by chromosome 02 (11). For
validation of the in silico mined polymorphic SSRs, F2 mapping population was
developed from the Selfing of F1s derived from the cross between gynoecious line
(GY-2) and monoecious line (CO-1). Gynoecy exhibited single recessive inheritance
in the F2 mapping population. Thirty-one polymorphic gSSRs and 06 eSSRs were
selected based on their ability to resolve on agarose gels. Among the 37 SSR markers
tested, none of them exhibited polymorphism between the parents on the 4% agarose
gel. On capillary electrophoresis, 03 markers (BG_gSSR253, BG_gSSR3902 and
BG_gSSR2363) exhibited polymorphism between gynoecious line and monoecious
lines. However, none of the 3 markers exhibited consistent polymorphism among the
pooled DNA of 10 each of monoecious and gynoecious F2 lines. In addition to SSR
markers, previously reported gynoecy governing chromosomal regions of bitter gourd
were sequenced for identification of SNPs between gynoecious and monoecious
parents. None of the five SNPs exhibited polymorphism between the monoecious and
gynoecious parents. Nonetheless, SSRs developed in this study are novel and may be
helpful in mapping gynoecy in other mapping populations.