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5.4.3 Maize Phenotypic and molecular analyses of maize landrace accessions in India

Evaluation of agronomic performance of a set of 125 landrace accessions of maize from diverse agroecologies in India was undertaken through multi-location trials at Tadong (Sikkim), Almora (Uttarakhand), Bajaura (HP), Delhi and Hyderabad (AP) during the last two years (2006 and 2007).

The biplot analysis demonstrated that landrace accessions could exhibit both location-specific and broader adaptation.

Twenty highly promising accessions were identified based on their performance for yield attributes and flowering behaviour.

Molecular characterization of 91 landrace accessions of maize in India was completed using 42 fluorescent-labeled SSR markers with allele resolution through MegaBACE DNA Sequencer, and estimation of allele frequencies using FreqsR.

Analysis using POWERMARKER revealed a total of 656 alleles, with a mean of 16 alleles per locus, and high mean PIC value of 0.61.

Identification of 187 unique/private SSR alleles led to clear differentiation of the accessions. The study also revealed six highly frequent/major SSR alleles at seven loci (phi062, umc1266, umc1367, phi090, phi014, phi112 and umc2250) with individual frequencies >0.70. These SSR loci were reported to tag specific genes/QTLs associated with some important traits such as disease resistance, anthesis-silking interval (ASI) and nutritional quality. Thus, it is clear that the chromosomal regions harboring these specific SSRs are not selectively neutral in landraces.

Analysis of various population genetic parameters revealed significant population substructure in the Indian maize landraces. Variation within populations was the highest (61.63%), followed by variation among the populations (37.51%) and variation among the groups (0.86%).

Cluster analysis of SSR allele data showed eight clusters, which largely matched with the patterns derived through analysis of performance data. A ‘core set’ of 15 landrace accessions was identified using ‘advanced M (Maximation) strategy’ that captured the phenotypic and molecular diversity among 91 accessions.

The study led to the clear differentiation of ‘Sikkim Primitives’ (distributed mainly in Sikkim with a few accessions from neighbouring NEH states) from the rest of the accessions, even within Sikkim. Eight ‘pools’, including

DNA sequencer image of SSR alleles (reflected as distinct peaks) in a specific accession

three based on promising NEH and non-NEH accessions (for yield traits) and five ‘Specialty corn’ pools (including baby corn, pop corn, and high carotenoid) were constituted (presently in C3/C4 stage). Analysis of genetic structure of diverse maize inbred lines in India

A detailed analysis of genetic structure in a panel of 69 maize inbred lines, including 58 lines developed under the All-India Coordinated Maize Improvement Project (since 1960s) and 11 genotypes from CIMMYT, was undertaken using microsatellite marker dataset. The inbred lines were subdivided into five genetic clusters (highly congruent with the pedigree data) using a model-based approach with the software package STRUCTURE; some inbred lines showed high genetic sub-structure.

To aid the breeders in choosing a diverse and representative collection, a ‘core set’ of inbred lines that captured the maximal SSR allelic diversity was identified.

This is the first study in India providing a comprehensive understanding of the genetic sub-structure within maize inbred lines that have been used by various breeding programmes across the country. Allele mining in important genes controlling prolificacy and nutritional quality in maize

Allele mining for teosinte branched-1 (tb1) gene that controls prolificacy was undertaken on seven genotypes from a panel of 48 genotypes (including ‘Sikkim Primitives’ and a set of teosinte accessions). Preliminary analysis indicated significant nucleotide diversity in the tb1 alleles of the prolific ‘Sikkim Primitives’ compared to that in the tb1 alleles of normal maize.

Allele mining for Yellow1 (Y1) gene influencing carotenoid biosynthesis in the maize endosperm was undertaken on 20 genotypes (in three replicates) from a panel of 48 maize genotypes (including 14 promising lines identified for carotenoid content by CIMMYT, Mexico). Two gene- specific primer pairs for amplifying overlapping target regions of the 5′ UTR of the Y1 gene were successfully designed, and the PCR-amplified products were purified and sequenced. Molecular marker-assisted breeding for maize improvement

Gene pyramiding. Gene pyramiding for resistance to Turcicum leaf blight (TLB) and Polysora rust (PR) is being undertaken in eight recurrent parents (CM137, CM138, CM139, CM140, CM145, CM212, CM150, CM151).

Evaluation of disease reaction responses of BC2F1 at UAS-ARS, Nagenahalli (against TLB and PR) and VPKAS, Almora (against TLB) during kharif 2007 led to the identification of highly promising genotypes. These were simultaneously screened using molecular markers, and selected plants were selfed to generate BC2F2 families.

Promising BC2F2 progenies are being advanced to BC2F3 at the Maize Winter Nursery, Hyderabad (rabi 2007- 08) after MAS, for fixation of resistance genes

Marker assisted transfer of opaque2. The objective of the project was to transfer of opaque2 (o2) gene into the six inbred lines (CM137, CM138, CM139, CM140, CM150, CM151) through molecular marker-assisted backcrossing, coupled with phenotypic selection for endosperm modifiers.

MAS (both foreground and background selections using SSR markers) was undertaken on BC2F1 populations, and BC2F2 progenies were generated during kharif 2007 at IARI, New Delhi.

Besides phenotypic selection for the recurrent parent phenotype, biochemical analysis of the BC2F2 progenies was also undertaken; the results clearly revealed the nutritional superiority (3-5-fold improvement in tryptophan content) in the lines generated.

MAS for o2/o2 homozygotes was undertaken on BC2F2 progenies during rabi 2007-08 (Maize Winter Nursery, Hyderabad) and the identified plants were selfed for deriving BC2F3 lines.

Transgenics in crop plants. Analysis of drought stress responses of 24 selected CIMMYT-DTP inbred lines was undertaken during kharif 2007 at DMR rain-out shelter, New Delhi, leading to the identification of promising drought tolerant genotypes.

RNA isolations were done from different tissues (silk, ear leaf and tassel leaf) of five genotypes from ‘control’ and

‘drought stress blocks’ during kharif 2007 at IARI, New Delhi. The experiment was repeated at ICRISAT, Hyderabad, during rabi 2007-08. The isolated RNA is being used for transcriptome profiling using maize gene chips.

In silico mining of 40,083 drought stress responsive maize ESTs in GenBank database led to the identification of 1162 EST-SSR markers. The EST sequences were further clustered to reduce redundancy, and consensus sequences were identified through sequence cleaning, repeat masking, vector masking and organelle masking. The consensus sequences were then sorted out based on the microsatellite

repeat motif and sequence length, and primers were designed for an initial set of ~70 EST-SSRs.

Transcriptome profiling for downy mildew stress resistance in maize was undertaken for NAI 116, a highly resistant maize inbred line using maize gene chips; for this, RNA was isolated from a field experiment undertaken at UAS-RRS, Mandya during kharif 2007.

Allele mining is being undertaken for several genes, including tb1, Y1 and su1, which includes both phenotyping for specific target traits and sequencing of PCR-amplified alleles for SNP/haplotype detection.

Characterization of global maize populations:

Tracking the maize migration routes from the centre of origin. Molecular characterization of the 288 global maize landraces (including accessions of the wild relative of maize, teosintes) were completed using five fluorescent-labelled microsatellite/SSR markers (phi076, phi075, phi299852, phi014 and umc2047).

The study has led to the identification of several unique alleles among the Indian maize landraces as compared to those among populations/landraces from other Asian countries.

Teosinte is the closest related species of maize and includes maize’s progenitor, Zea mays ssp. parviglumus.

Maize biofortification. For analysis of kernel micronutrients (Fe and Zn) content, a set of 60 maize genotypes was evaluated at IARI, New Delhi, during kharif 2007. Data analyses (for kernel Fe and Zn), led to the identification of a set of highly promising maize genotypes (including HP 2, BAJIM 19, V 336, IML 119, IML 288, IML 434 and IML 467).

Twenty-four phenotypically contrasting inbred lines were analysed using 40 microsatellite/SSR markers covering the maize genome leading to the detection of 178 SSR alleles, at an average of 4.45 alleles per locus. The SSR analysis aided in the selection of specific crosses for generation of different mapping populations for undertaking QTL mapping of kernel Fe and Zn concentrations in maize.

Determining EDVs in maize. Twenty-one maize lines, including different initial varieties (IVs), putative essentially derived varieties (EDVs) and independently derived checks, were genotyped using 40 fluorecent-labelled SSR markers (with allele resolution using MegaBACE DNA Sequencer 1000) during the reported period. The data will be statistically analyzed for determination of thresholds for ascertaining EDVs in maize. Molecular breeding for crop improvement in maize

Three promising NEH and non-NEH pools were constituted and advanced to Cycle 4 during rabi 2007-08 from a set of landrace accessions of these regions, through multi-location trials during 2006-2007.

Five specialty corn pools of baby corn, popcorn and high carotenoid genotypes were advanced to Cycle 3 during rabi 2007-08 at the Winter Maize Nursery, Hyderabad. Two baby corn pools, one of early maturity and the other of full- season maturity group, are presently in Cycle 3. Also, one highly promising pop corn pool and two high carotenoid pools are at Cycle 3/Cycle 4.

Synthesis of a set of specialty corn inbred lines (S3) was also undertaken during the reported period. Eleven promising pop corn inbred lines, four baby corn and five high carotenoid inbred lines were identified during rabi 2007- 08. These are being advanced. A set of other specialty corn inbred lines (S2) was also advanced during kharif 2007.

A set of promising baby corn, pop corn, high carotenoid, sugary inbred lines/populations and QPM genotypes obtained from CIMMYT, Mexico and different breeding centres in India for utilization in the breeding programmes were maintained.

NEH semi-flint pool (Cycle 3) (left), and representative ears (right)

Non-NEH high carotenoid pool (Cycle 3) and selected ears from the pool Fingerprinting and molecular diversity analysis

Sixteen popular inbred lines, 8 released and 8 experimental, were analyzed using 24 microsatellites or Simple Sequence Repeats (SSR) markers. The study revealed 28 rare alleles, out of which 9 were unique to some of the inbred lines. The average polymorphism information content (PIC) and discrimination rate (DR) were 0.39 and 0.61, respectively. Genetic similarity expressed as Jaccard’s coefficient varied from 0.23 to 0.68 with an average of 0.41.

Cluster analysis using unweighted pair group method with arithmetic averages (UPGMA) suggested five clusters. All the genotypes studied could be clearly distinguished with a minimum set of three markers with high PIC and DR. Efforts towards characterization, utilization and introgression of sweet corn trait

Development of experimental hybrids involving 12 sweet corn parental lines was initiated involving parental lines with wider genetic base, including from the crosses between normal x sweet corn combinations. The introgression of sweetness trait into elite field corn inbred lines was taken up in a total of 32 crosses from four sources of sweet corn. Characterization and transfer of disease resistance

Banded leaf and sheath blight (BLSB). On the basis of multiple screening against BLSB pathogen, seven maize inbred lines showing a range of response from 2.5 to 4.5 in a rating of 1-5 were identified. Screening and genetic analysis against biotic stresses

Response of different maize genotypes and their crosses was analyzed against BLSB (banded leaf and sheath blight) under artificial inoculation conditions. Eight elite parental

lines were crossed with the registered genetic stocks carrying resistance to MLB (Maydis leaf blight) for the purpose of introgression of resistance.