Allelic variation and haplotype diversity of Matrilineal (MTL) gene governing in vivo maternal haploid induction in maize.

Diverse haploid inducer lines with > 6% of haploid induction rate are now routinely used to develop doubled haploid lines. Though MTL gene regulates haploid induction, its molecular characterization and haplotype analysis in maize and its related species have not been undertaken so far. In the present study, the entire 1812 bp long MTL gene was sequenced among two mutant and eight wild-type inbreds. A 4 bp insertion differentiated the mutant from the wild-type allele. Sequence analysis further revealed 103 polymorphic sites including 38 InDels and 65 SNPs. A total of 15 conserved regions were detected, of which exon-4 was the most conserved. Ten gene-based markers specific to MTL revealed the presence of 40 haplotypes among diverse 48 inbreds of exotic and indigenous origin. It generated 20 alleles with an average of two alleles per locus. The mean polymorphic information content was 0.3247 with mean gene diversity of 0.4135. A total of 15 paralogous sequences of MTL were detected in maize genome with 3-7 exons. Maize MTL proteins of both wild-type and mutant were non-polar in nature, and they possessed four domains. R1-nj-based haploid inducer (HI) lines viz., Pusa-HI-101 and Pusa-HI-102 had an average haploid induction rate of 8.45 ± 0.96% and 10.46 ± 1.15%, respectively. Lines wild-type MTL gene did not generate any haploid. In comparison with 27 orthologues of 21 grass species, maize MTL gene had the closest ancestry with Saccharum spontaneum and Sorghum. The information generated here assumes great significance in understanding the diversity of MTL gene and presence of paralogues and orthologues. This is the first report on haplotype analysis and molecular characterization of MTL gene in maize and related grass species. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01456-3.

Variation in anthocyanin pigmentation by R1-navajo gene, development and validation of breeder-friendly markers specific to C1-Inhibitor locus for in-vivo haploid production in maize.

BACKGROUND: In-vivo maternal haploids serve as the basis of doubled haploid (DH) breeding in maize. R1-navajo (R1-nj) gene governing anthocyanin colouration in the endosperm and embryo is widely used to identify haploid seeds. However, the expression of R1-nj depends on genetic-background of source-germplasm used for deriving DH-lines. Further, presence of C1-Inhibitor (C1-I) gene suppresses the expression of R1-nj, thus makes the selection of haploids difficult. METHODS: In the present study, 178 subtropically-adapted maize inbreds were crossed with two R1-nj donors 'that do not have haploid induction genes'. Of these, 76.4% inbreds developed purple colour in endosperm, while 23.6% did not show any colouration. In case of scutellum, 62.9% inbreds possessed colour and 37.1% were colourless. The anthocyanin intensity varied greatly, with 19.66% and 42.98% inbreds displayed the least intensity, while 16.85% and 0.84% inbreds showed the highest intensity in endosperm and scutellum, respectively. Two C1-I specific breeder-friendly markers (MGU-CI-InDel8 and MGU-C1-SNP1) covering (i) 8 bp InDel and (ii) A to G SNP, respectively, were developed. MGU-CI-InDel8 and MGU-C1-SNP1 markers predicted presence of C1-I allele with 92.9% and 84.7% effectiveness, respectively. However, when both markers were considered together, they provided 100% effectiveness. CONCLUSIONS: These markers of C1-I gene would help in saving valuable resources and time during haploid induction in maize. The information generated here assume great significance in DH breeding of maize.

Molecular analysis of mutant granule-bound starch synthase-I (waxy1) gene in diverse waxy maize inbreds.

Waxy corn is popular beacuse of its high amylopectin due to mutation in granule-bound starch synthase-I or Waxy1 (Wx1) gene. Here, we characterized the wx1 allele among 24 diverse waxy inbreds using gene-based markers. A total of 29 alleles with average of 1.81 alleles/locus were observed. Major allele frequency varied from 0.42 to 1.00, with mean of 0.74. The polymorphism information content ranged from 0.00 to 0.56 (average 0.24). Three simple sequence repeat markers, viz., phi027, phi022 and phi061 were more polymorphic in the study. The mean heterozygosity was 0.04, which indicated attainment of higher levels of homozygosity. Dissimilarity coefficient varied from 0.00 to 0.90 with average of 0.51. Seventeen diverse haplotypes of wx1 allele were observed that was consistent with the pedigree. Cluster analyses grouped 24 genotypes into two main clusters each having sub-clusters. The information generated here possesses great potential for improvement of high amylopectin in maize through marker-assisted selection. This is the first report of molecular dissection of wx1 gene among the novel waxy inbreds developed in India.