(a) Explain briefly the process and importance of the following : 5+5+5=15 (i) Male sterility and heterosis breeding. 5 (ii)…

(a)(i) Male sterility and heterosis breeding

Heterosis (hybrid vigour) is the superiority of an F1 over its parents in yield, growth or vigour. It is explained by the dominance hypothesis (favourable dominant alleles from both parents mask each other's harmful recessives) and by overdominance (the heterozygote itself is superior). It is largest between genetically distant inbreds, and it is lost in F2 by segregation, so fresh F1 seed must be produced every season. On a large scale that is practical only if the female parent cannot self-pollinate. Male sterility, the failure to produce functional pollen or anthers, removes the need for hand emasculation.

• Genic male sterility (GMS): caused by recessive nuclear genes (ms). Fertile sibs must be rogued out, so its use is limited.
• Cytoplasmic male sterility (CMS): caused by chimeric mitochondrial genes, such as T-urf13 in maize T-cytoplasm. It is useful where the product is vegetative, as in onion and carrot.

Cytoplasmic–genic male sterility (CGMS): sterile cytoplasm plus nuclear restorer (Rf) genes. This gives the three-line system:

• The A-line (male-sterile) is maintained by crossing with the B-line (same nucleus, normal cytoplasm).
• The hybrid is A × R, where the R-line carries Rf, so the F1 is fertile and sets grain.
• Environment-sensitive GMS: thermo- or photoperiod-sensitive GMS (TGMS/PGMS) gives the simpler two-line hybrid rice system.

Examples and importance

• Pearl millet: India's first hybrid, HB-1 (1965), used Tift 23A.
• Rice: Chinese hybrid rice (Yuan Longping) used wild-abortive (WA) cytoplasm, and India released its first rice hybrids in 1994.
• Other crops: hybrids of sorghum (CSH series), sunflower and cotton, and seed-parent lines in vegetables.
• Caution: maize hybrids yield much more than open-pollinated varieties. However, the 1970 US southern corn leaf blight epidemic struck because most hybrids shared T-cytoplasm, which shows the risk of a narrow cytoplasmic base.

(a)(ii) Molecular basis of the cell cycle

The cycle (G1 → S → G2 → M) is driven by cyclin-dependent kinases (CDKs). They are active only when bound to a cyclin, whose level rises and falls in each cycle. Hartwell, Hunt and Nurse shared the 2001 Nobel Prize for this discovery.

• G1 → S: growth signals induce cyclin D. Cyclin D–CDK4/6 (in plants, CDKA with cyclin D) phosphorylates the retinoblastoma protein (Rb/RBR). This releases E2F, which switches on S-phase genes. Cyclin E/A–CDK2 then starts DNA replication.
• G2 → M: cyclin B–CDK1, called maturation (M-phase) promoting factor (MPF), drives chromosome condensation, nuclear-envelope breakdown and spindle formation. It is activated by the phosphatase Cdc25 when the inhibitory kinase Wee1 is overcome.
• Exit from M: the anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase activated by Cdc20. It tags securin and cyclin B for destruction by the proteasome. Separase then cuts cohesin, the sister chromatids separate, and CDK activity falls so the cell can divide.

Checkpoints:

• G1/S: DNA damage activates ATM/ATR and p53, which induces the CDK inhibitor p21.
• G2/M: unreplicated or damaged DNA blocks MPF.
• Spindle assembly checkpoint: Mad/Bub proteins hold back APC/C until every kinetochore is attached.
• Plant-specific features: plants have extra CDKB types, and KRP inhibitors instead of p21.

Loss of checkpoints (for example p53 or Rb mutations) causes uncontrolled division in cancer. In plants, cell-cycle genes control organ size and endoreduplication.

(a)(iii) Gene silencing

Gene silencing switches off a gene's expression without changing its DNA sequence. This is unlike a knockout, which destroys the gene.

• Transcriptional gene silencing (TGS): DNA methylation of the promoter and repressive histone marks stop transcription. In plants this is directed by 24-nt siRNAs through RNA-directed DNA methylation (RdDM), and it keeps transposons silent.
• Post-transcriptional gene silencing (PTGS) or RNA interference (RNAi): double-stranded RNA is cut by Dicer (DCL in plants) into 21–24-nt siRNAs, or folded precursors are cut into miRNAs. These load onto Argonaute in the RISC complex and guide it to complementary mRNA, which is cleaved or not translated.
• Discovery: co-suppression was first seen in petunia, where extra copies of the chalcone synthase gene gave white flowers (Napoli and Jorgensen, 1990). The RNAi mechanism was shown in C. elegans by Fire and Mello (1998; Nobel Prize 2006).

Importance

• Natural role: it defends against viruses and transposons, and miRNAs regulate development (for example, miR165/166 and leaf polarity).
• Research tool: RNAi and virus-induced gene silencing (VIGS) knock genes down to reveal their function.

Crop improvement:

• Flavr Savr tomato: antisense silencing of polygalacturonase for slower softening.
• Rainbow papaya: resistant to papaya ringspot virus through coat-protein-mediated silencing.
• Arctic apple: non-browning, by silencing polyphenol oxidase.
• Cottonseed: gossypol reduced in the seed only, by silencing δ-cadinene synthase.
• Host-induced gene silencing against nematodes and fungi.

(b) Methods of gene mapping, and how molecular maps help evaluate gene function

1. Genetic (linkage) mapping. Gene order and distance are found from the frequency of recombination at meiosis.

• Classical crosses: two-point and three-point test crosses (Sturtevant, 1913). The least frequent class gives gene order, and 1% recombination = 1 centimorgan (cM). Mapping functions (Haldane, Kosambi) correct for double crossovers and interference.

Molecular markers make dense maps:

• Hybridisation-based: RFLP.
• PCR-based: RAPD, AFLP, SSR (microsatellites).
• High-throughput: SNPs by arrays or genotyping-by-sequencing.
• Markers are neutral, numerous and unaffected by environment.
• Mapping populations: F2, backcross, recombinant inbred lines (RILs), doubled haploids and near-isogenic lines. Software such as MAPMAKER or JoinMap builds the linkage groups.
• QTL mapping: interval mapping (LOD score) locates genes for quantitative traits.
• Association mapping (GWAS): this uses historical recombination in diverse germplasm and gives finer resolution.

2. Cytogenetic mapping. Genes are assigned to chromosomes or arms using aneuploids (monosomics and nullisomics in wheat), deletion stocks, translocations, and fluorescence in situ hybridisation (FISH) on chromosomes.

3. Physical mapping. Distance is measured in base pairs.

• Restriction mapping of cloned DNA.
• Contig maps: overlapping BAC/YAC clones are ordered by fingerprinting (FPC) and anchored to markers.
• Radiation-hybrid and optical maps (restriction sites on single stretched DNA molecules).
• The relation between cM and kb varies along the chromosome, because recombination is suppressed near centromeres.

4. Sequence map. This is the ultimate physical map: the whole genome sequence with annotated genes. Examples are Arabidopsis (2000) and rice (IRGSP, 2005, with Indian participation). Pan-genomes now capture variation among cultivars.

How molecular maps help evaluate gene function

Map-based (positional) cloning:

• A trait is mapped between flanking markers and fine-mapped in large populations.
• The interval is found on the physical or sequence map, and candidate genes are tested by complementation, knockout (T-DNA, CRISPR) or expression studies.
• **Rice Sub1A:** the submergence-tolerance gene was cloned in 2006 and introduced into Swarna to give Swarna-Sub1.
• **Tomato fw2.2:** the first cloned fruit-weight QTL.
• Resistance genes: rice blast (Pi-ta) and bacterial blight (Xa21) genes were isolated this way.
• QTL and eQTL mapping: these link quantitative variation, and the expression level of genes, to specific regions. They separate major from minor genes and reveal pleiotropy.
• Comparative mapping (synteny): gene order is conserved among grasses (rice, wheat, maize, sorghum). A gene cloned in rice predicts its orthologue in a large-genome crop such as wheat.
• Marker-assisted selection and gene pyramiding: mapped genes can be combined without phenotyping each generation. Examples are xa5, xa13 and Xa21 in Improved Samba Mahsuri, and Sub1 varieties. The markers themselves confirm the gene's effect in new backgrounds.
• Reverse genetics: mutants and insertion lines are anchored to the map, which links sequence to phenotype. Where a gene lies, and what lies near it, also helps predict linkage drag.

(c) Apomixis and its use in plant breeding

Apomixis (agamospermy) is the formation of seeds without meiosis and fertilisation, so the embryo is a genetic clone of the mother plant (Winkler's usage). It is distinct from parthenocarpy, which gives seedless fruit, and from vegetative propagation.

Types

Gametophytic apomixis: an unreduced (2n) embryo sac forms, and its egg develops parthenogenetically.

• Diplospory: the embryo sac comes from the megaspore mother cell after failed or no meiosis, as in Taraxacum and Antennaria.
• Apospory: the embryo sac comes from a somatic nucellar cell, as in Hieracium, Pennisetum/Cenchrus and Panicum maximum (Guinea grass).
• Many aposporous grasses are pseudogamous: pollination is still needed to fertilise the central cell and form endosperm.
• Sporophytic apomixis (adventive or nucellar embryony): embryos bud directly from nucellus or integument cells, beside a sexual embryo. This causes polyembryony in Citrus, mango (polyembryonic types) and Opuntia.
• Genetic control: apomixis is usually dominant and often inherited as a single locus, the apospory-specific genomic region (ASGR) of Pennisetum squamulatum. An ASGR-BABY BOOM-like gene from this region induces parthenogenesis (Conner et al., 2015).

Uses in plant breeding

• Fixing heterosis: an apomictic F1 hybrid would breed true. Farmers could save hybrid seed every year without losing vigour, and small-farm crops could get hybrids without a costly seed industry. This is the main goal.
• Fixing any genotype: superior genotypes, including polyploids and odd-chromosome types, can be fixed at once and multiplied through seed rather than cuttings.

Uniform, disease-free stock:

• Nucellar seedlings of Citrus (rough lemon, Rangpur lime) give true-to-type rootstocks.
• They are free of most viruses, since viruses generally do not pass through seed.

Apomictic cultivars of forage and turf grasses:

• Kentucky bluegrass (Poa pratensis), buffel grass (Cenchrus ciliaris) and Guinea grass cultivars are propagated true from seed.
• In India, IGFRI Jhansi works on apomictic forage grasses.

Synthetic apomixis:

• MiMe + BBM1 (Khanday et al., 2019): the MiMe mutations (pair1, rec8, osd1) replace meiosis with mitosis. Together with expression of BABY BOOM1 in the egg cell, they gave clonal seeds of hybrid rice.
• MiMe + MTL (Wang et al., 2019): combining MiMe with MTL knockout gave the same result.
• Later work has raised the frequency of clonal seed.

Limitations. Obligate apomicts cannot be recombined further. Facultative apomixis is unstable. Endosperm needs a correct maternal : paternal genome ratio. The trait is linked to polyploidy and to suppressed recombination in wild donors. So breeding uses sexual lines to create variation and apomixis to fix the best combination.