A multiple qtl-seq strategy delineates potential genomic loci governing flowering time in chickpea
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A total of 592486 SNPs (with a mean map-density of 0.56
kb) were found polymorphic between early (ICC 4958 and
EDTFB) and late (ICC 8261 and LDTFB) flowering mapping
parents and bulks as per their congruent physical locations (bp)
on the reference desi genome (pseudomolecule) (Table 2 and
Supplementary Tables S2, S3). These genome resequencing-led
SNPs were subsequently utilized for QTL-seq analysis.
The SNP-index of individual SNPs exhibiting differentiation
between early (ICC 4958 and EDTFB) and late (ICC 8261 and
LDTFB) flowering mapping parents and bulks was estimated.
The mean SNP-index (within a 1-kb sliding window and
10 Mb genomic interval) as well as 1 (SNP-index) of
EDTFB and LDTFB were measured and further plotted across
chromosomes as per aforesaid methods (Figure 2B). This
essentially detected two major genomic regions (CaqbDTF4.1:
45600294 to 46991993 bp and CaqbDTF4.2: 26500027 to
27407090 bp) on chromosome 4 revealing the mean SNP-index
of ≥ 0.8 in EDTFB and ≤ 0.2 in LDTFB (Figures 2B, 3C,D). The
accuracy of these major genomic regions underlying DTF QTLs
was ascertained by a valid 99% 1 (SNP-index) significance level.
The comprehensive analysis of these DTF QTL genomic regions
inferred the occurrence of majority of the SNP alleles derived
from parents (ICC 4958 and ICC 8261) in early and late flowering
mapping individuals forming the EDTFB and LDTFB bulks,
respectively. Overall, the QTL-seq in an intra-specific mapping
population (ICC 4958 × ICC 8261) identified two major DTF
QTLs- CaqbDTF4.1 and CaqbDTF4.2- at the 907.1 kb [26500027
(SNP_1B) to 27407090 (SNP_2B) bp with a 1 (SNP-index): 0.9]
and 1.39 Mb [45600294 (SNP_3B) to 46991993 (SNP_4B) bp
with a 1 (SNP-index): 0.8] genomic intervals, respectively, on
chickpea chromosome 4 (Figure 3B).
The detailed structural annotation of 7302 and 3177 SNPs
at CaqbDTF4.1 and CaqbDTF4.2, respectively, revealed the
occurrence of 32.5 to 49.1% of SNPs in the genes and rest in the
intergenic regions (Supplementary Table S4). The gene-derived
SNPs included highest and lowest proportion of 73.8–74.6%
and 1.2–1.5% SNPs in the DRRs and URRs, respectively.
The coding SNPs included the 52.1–54.7% synonymous and
45.3–47.9% non-synonymous (missense and nonsense) SNPs
(Supplementary Table S4). The allelic variants of SNPs covering
the QTL-seq led major DTF QTLs (CaqbDF4.1 and CaqbDF4.2)
were validated by resequencing of PCR fragments amplified
from the parents (ICC 4958 and ICC 8261) and mapping
individuals composing the EDTFB and LDTFB bulks. Like-wise,
we detected two major DTF QTLs on similar aforementioned
physical positions of chromosome 4 by the QTL-seq analysis
of EDTFB and LDTFB bulks constituted from a RIL mapping
population (ICC 4958 × ICC 8261) using the long- and shortday
photoperiod-based greenhouse DTF phenotyping data of
chickpea