(a) What are the different methods of DNA sequencing? Elaborate the shotgun sequencing method. (20 marks) (b) Describe the…

(a) Methods of DNA Sequencing and the Shotgun Method

DNA sequencing methods fall into three generations.

1. First generation: the Maxam–Gilbert chemical cleavage method (1977), in which end-labelled DNA is broken at specific bases by chemicals; and Sanger's dideoxy chain-termination method (1977), in which DNA polymerase copies the template and random incorporation of ddNTPs stops the chains. Automated Sanger sequencing with fluorescent ddNTPs and capillary electrophoresis gives reads of about 800–1,000 bases with very high accuracy but low throughput.
2. Second generation (next-generation sequencing, NGS): massively parallel sequencing of millions of amplified fragments, such as 454 pyrosequencing (light from released pyrophosphate), Illumina sequencing by synthesis with reversible terminators, and Ion Torrent semiconductor detection of released H⁺. Throughput is enormous and cost low, but reads are short (about 100–300 bases).
3. Third generation: single-molecule, real-time sequencing without amplification, namely PacBio SMRT sequencing and Oxford Nanopore sequencing (reading changes in current as DNA passes through a protein pore). Reads of tens to hundreds of kilobases resolve repeats and complex regions.

Shotgun sequencing determines the sequence of a long DNA molecule or a whole genome by breaking it at random into many small overlapping fragments, sequencing them, and assembling them by computer. Sanger's group used it for bacteriophage λ, and Fleischmann, Venter and colleagues (1995) sequenced the first free-living organism, Haemophilus influenzae, by whole-genome shotgun sequencing. Celera then applied it to Drosophila (2000) and the human genome (2001). The steps are:

1. Random fragmentation: genomic DNA is sheared by sonication, nebulisation or enzymes into random fragments of defined size.
2. Library construction: the fragments are cloned into plasmid vectors (e.g., 2 kb and 10 kb inserts), with fosmid or BAC libraries for long-range links, or ligated to adapters for NGS platforms.
3. Sequencing: clones or fragments are read from both ends (paired-end or mate-pair reads) to a depth of about 8–10× (much higher for NGS), so that every base is covered several times.
4. Assembly: software (e.g., Phrap or Celera Assembler for Sanger reads; de Bruijn graph assemblers for short reads) detects overlaps between reads and joins them into contigs; paired-end information orders and orients contigs into scaffolds.
5. Finishing: gaps are closed by directed sequencing or PCR, and the assembly is checked against genetic or physical maps.

Compared with hierarchical (clone-by-clone) sequencing, in which mapped BAC clones are sequenced one by one, whole-genome shotgun is faster and cheaper but assembles repeat-rich plant genomes less accurately; long reads now largely overcome this. Indian examples include draft pigeonpea genomes published in 2012 by an ICRISAT-led international consortium and by an ICAR-led Indian consortium, the chickpea genome (2013), and India's contribution to the rice genome (IRGSP).

(b) Linkage and Mendel's Second Law

Linkage is the tendency of genes located close together on the same chromosome to be inherited together, so that parental combinations appear in the progeny more often than new (recombinant) combinations. The genes of one chromosome form a linkage group, and the number of linkage groups equals the haploid chromosome number (for example, 10 in maize and 4 in Drosophila melanogaster).

Examples:

1. **Sweet pea (Lathyrus odoratus): Bateson and Punnett (1906) crossed plants with purple flowers and long pollen (PPLL) with plants having red flowers and round pollen (ppll). The F₂ did not give 9:3:3:1; the parental types were in excess. They called this coupling when the dominant alleles came from one parent and repulsion** when they came from different parents.
2. ***Drosophila*: T.H. Morgan (1910–11) showed that body colour and wing length (grey, long × black, vestigial) are linked. When F₁ males were test-crossed, only the parental types appeared (complete linkage**, since male Drosophila show no crossing over). F₁ females gave mostly parental types with about 17% recombinants (incomplete linkage). Morgan explained this by the physical location of genes on chromosomes and crossing over.
3. Maize: the genes for coloured aleurone (C) and shrunken endosperm (sh) on chromosome 9 show only about 3% recombination (Hutchison, 1922).

Why linkage is an exception to Mendel's second law: Mendel's law of independent assortment states that alleles of different genes segregate into gametes independently, so a dihybrid produces four kinds of gametes in equal proportion (1:1:1:1) and the F₂ shows 9:3:3:1. This holds only for genes on different chromosomes, or very far apart on the same chromosome. Linked genes travel together on one chromosome and are separated only by crossing over. The dihybrid therefore produces more parental than recombinant gametes, the test-cross ratio departs from 1:1:1:1, and the F₂ ratio departs from 9:3:3:1. The recombination frequency (1% = 1 map unit or centimorgan) measures the strength of linkage and is used to build linkage maps (Sturtevant, 1913). Mendel's seven characters in pea happened to show independent assortment; some were on the same chromosome but so far apart that they behaved as unlinked.

(c) Structure and Behaviour of B Chromosomes in Plants

B chromosomes (supernumerary or accessory chromosomes) are dispensable chromosomes present in addition to the standard (A) complement in some individuals of a population. They are not needed for normal growth and reproduction. They were first described in insects (Wilson, 1907), and in maize by Kuwada (1915) and Randolph (1928). They occur in well over a thousand plant species, e.g., maize, rye, Aegilops speltoides, Allium, Lolium and Brachycome, and their number varies between plants and between populations.

Structure:

1. They are usually smaller than A chromosomes and do not pair with them; for example, the maize B is acrocentric with a small short arm and a long arm with heterochromatic blocks.
2. They are largely heterochromatic and rich in repetitive DNA (satellite repeats, transposable elements); some repeats, such as the ZmBs repeat at the maize B centromere and the D1100/E3900 families in rye, are B-specific.
3. They contain few active genes, many of them copies of A-chromosome sequences, and organelle-derived DNA; a few are transcribed.
4. They have a functional centromere, which is sometimes misdivided to form isochromosomes and telocentrics.

Behaviour:

1. Non-Mendelian inheritance and accumulation (drive): Bs are transmitted at rates above 50%. In maize, the B undergoes non-disjunction at the second pollen mitosis, so one sperm gets two Bs and the other none; the B-carrying sperm then preferentially fertilises the egg (about 60–70%). In rye, non-disjunction occurs at the first pollen mitosis (and at the first egg mitosis), the Bs passing to the generative nucleus.
2. Meiotic irregularity: Bs pair only among themselves, forming bivalents, trivalents or univalents. Univalents often lag at anaphase and are lost, which partly balances their accumulation.
3. Effects on the host: low numbers have little effect, but high numbers reduce vigour, fertility and seed set. In maize they increase recombination in A chromosomes, and in Lolium and Ae. speltoides hybrids they suppress homoeologous pairing.
4. Population dynamics: B frequency reflects a balance between drive and harmful effects, which is why Bs are regarded as selfish genetic elements. They are useful for studying centromere function and genome evolution, and as vectors in chromosome engineering (e.g., maize B mini-chromosomes).