Development and Integration of Genome-Wide Polymorphic Microsatellite Markers onto a Reference Linkage Map for Constructing a High-Density Genetic Map of Chickpea

Abstract

The identification of informative in silico polymorphic genomic and genic microsatellite markers by comparing the genome and transcriptome sequences of crop genotypes is a rapid, cost-effective and non-laborious approach for large-scale marker validation and genotyping applications, including construction of high-density genetic maps. We designed 1494 markers, including 1016 genomic and 478 transcript-derived microsatellite markers showing in-silico fragment length polymorphism between two parental genotypes (Cicer arietinum ICC4958 and C. reticulatum PI489777) of an inter-specific reference mapping population. High amplification efficiency (87%), experimental validation success rate (81%) and polymorphic potential (55%) of these microsatellite markers suggest their effective use in various applications of chickpea genetics and breeding. Intra-specific polymorphic potential (48%) detected by microsatellite markers in 22 desi and kabuli chickpea genotypes was lower than inter-specific polymorphic potential (59%). An advanced, high-density, integrated and inter-specific chickpea genetic map (ICC4958 x PI489777) having 1697 map positions spanning 1061.16 cM with an average inter-marker distance of 0.625 cM was constructed by assigning 634 novel informative transcript-derived and genomic microsatellite markers on eight linkage groups (LGs) of our prior documented, 1063 marker-based genetic map. The constructed genome map identified 88, including four major (7-23 cM) longest high-resolution genomic regions on LGs 3, 5 and 8, where the maximum number of novel genomic and genic microsatellite markers were specifically clustered within 1 cM genetic distance. It was for the first time in chickpea that in silico FLP analysis at genome-wide level was carried out and such a large number of microsatellite markers were identified, experimentally validated and further used in genetic mapping. To best of our knowledge, in the presently constructed genetic map, we mapped highest number of new sequence-based robust microsatellite markers (634) which is an advancement over the previously documented (~300 markers) inter-specific genetic maps. This advanced high-density map will serve as a foundation for large-scale marker validation and genotyping applications, including identification and targeted mapping of trait-specific genes/QTLs (quantitative trait loci) with sub-optimal use of resources and labour in chickpea.

Fig 1. Summary of genomic and transcript-derived in silico polymorphic microsatellite markers used in the present study.

Fig 2. Validation of a representative set of novel transcript-derived (I) and genomic (II) microsatellite markers showing in silico FLP between ICC4958 (1) and PI489777 (2) using the gel-based assay (I and II) and fluorescent dye labeled automated fragment analyzer (III and IV).

The fragment sizes (bp) of the amplified polymorphic alleles are indicated. The identities of markers (A: CaTMS616, B: CaTMS654, C: CaTMS715, D: CaTMS716, E: CaTMS561, F: CaTMS577, G: CaTMS783, H: CaTMS651, I: CaGMS1, J: CaGMS3, K: CaGMS13, L: CaGMS16, M: CaGMS18, N: CaGMS19, O: CaGMS24, P: CaGMS23, Q: CaGMS20, R: CaGMS41, S: CaGMS43 and T: CaGMS45) with their detailed information are provided in the S2 Table. The primers CaTMS606 and CaGMS40 were used for automated fragment analysis (III and IV). M: 50 bp DNA ladder size standard.

Fig 3. The sequencing of cloned amplicons from different perfect transcript-derived (I) (CaTMS1055) and compound genomic (II) (CaGMS1156) microsatellite markers showing FLP between ICC4958 and PI489777 and their multiple sequence alignment (C and D) validated the presence of expected microsatellite repeat-motifs which corresponded well with our in silico prediction of expansion and contraction of a number of microsatellite repeat-units.

The fragment sizes (bp) of the amplified polymorphic alleles are indicated. M: 50 bp DNA ladder size standard. The identities of two markers with their detailed information are provided in the S2 Table.

Fig 4. Allelic variations detected among 22 desi and kabuli genotypes along with controls (ICC4958 and PI489777) using a representative set of novel genomic (A) (CaGMS24) and transcript-derived (CaTMS561) (B) microsatellite markers in gel-based assay.

A maximum number of four polymorphic alleles were amplified by the genomic microsatellite markers, while transcript-derived microsatellite markers produced two alleles among genotypes. The fragment sizes (bp) of the amplified polymorphic alleles are indicated. The genotypes used are, 1: ICC4958, 2: PI489777, 3: Pusa362, 4: Himchana1, 5:ICCC4, 6: Dilaji, 7: JG11, 8: Bharathi, 9: BGD112, 10: Pusa256, 11: JG62, 12: Kranthi, 13: JAKI9218, 14:Swetha, 15: JGK3, 16: PKV Kabuli2, 17: Vihar, 18: LBeG7, 19: BG5023, 20: Pusa1088, 21: BGD1105, 22: PG515, 23: Pusa1108 and 24: JGK2. M: 50 bp DNA ladder size standard. The identities of two markers with their detailed information are provided in the S2 Table.

Fig 5. Segregation pattern of transcript-derived (A) (CaTMS651) and genomic (B) (CaGMS16) microsatellite markers in a representative set of mapping individuals of a RIL population derived from the inter-specific cross between ICC4958 and PI489777 along with parental genotypes.

The amplified microsatellite marker alleles are resolved using agarose gel-based assay and fluorescent dye-labeled automated fragment analyzer. The fragment sizes (bp) of the amplified parental polymorphic alleles are indicated. M: 50 bp DNA ladder size standard. *indicates the heterozygous alleles amplified by microsatellite markers which are further confirmed through automated fragment analysis (C). The identities of two markers with their detailed information are provided in the S2 Table.

Fig 6. An advanced inter-specific, high-resolution and integrated genetic map (ICC4958 x PI489777) of chickpea constructed by assigning 634 novel genomic and transcript-derived microsatellite markers on eight LGs of a previously reported similar 1063 marker-based genetic map (Gaur et al. [26]).

The genetic distance (cM) and identity of the marker loci integrated are indicated on the left and right side of eight LGs, respectively. The earlier reported markers are considered as anchor markers to define eight LGs. The LGs are specified with Arabic numerals on the upper-side corresponding with the genetic map as reported by Gaur et al. [26]. The newly integrated genomic and transcript-derived microsatellite markers in this study are highlighted with red colour. Markers designated as CaGMS represent Cicer arietinum genomic microsatellite markers, whereas CaTMS represent genic C. arietinum transcript-derived microsatellite markers.

Fig 7. Frequency distribution of novel genomic and transcript-derived microsatellite markers mapped within 1 cM genetic distance on eight LGs of an integrated and inter-specific chickpea genetic linkage map (ICC4958 x PI489777) identified four major high-resolution genomic regions on LG3, LG5 and LG8 with higher marker map density (1–50 markers within 1 cM genetic distance).

These four longest high-resolution genomic regions showed a persistent distribution of mapped markers up to 5–23 cM genetic distance. The hot spot regions (cM) on LG3, LG5 and LG8 showing higher map density of microsatellite markers are marked with boxes.