Plant Genome Project | Genetics

In this article we will discuss about the plant genome project.

The major revolution in the study of genomes of different species was

brought about due to the availability of recombinant DNA and PCR
technologies. These techniques helped in preparation of molecular
maps of many plant and animal genomes. The objective of genomic
research in any species is to sequence the whole genome and to
decipher functions of all the different coding and non-coding
sequences.

The technology for large-scale DNA sequencing has enable scientists

to undertake genome sequencing project in a realistic time scale. Since
the time of first large genome sequencing in bacteriophage in 1983,
the projects on different groups have been completed.

Some notable examples include the bacterium Escherichia coli, the

yeast Saccharomyces cerevisae, the weed Arabidopsis thaliana, the rice
Oryza sativa, the nematode Caenorhabditis elegans, the fruit fly
Drosophila melanogaster, the mouse Mus musculus, the primate
chimpanzee Pan troglodytes, the human Homo sapiens sapiens.

In crop science genomics has been promoted using Arabidopsis, a

weed plant found all over the world, easy to grow, short span of life
cycle, one of the smallest genome among dicot plants and Oryza sativa
(rice), one of the members from monocot having very simple genome
organisation and similarity with other major cereal plants.

For any genome sequencing programme, the following steps

are undertaken (Fig. 22.18):

1. Construction of Linkage Maps with Molecular Markers:

Different kinds of molecular markers like RFLP, RAPD,

microsatellites; AFLP, etc. are used to construct the actual physical
linkage map. The RFLP markers are then hybridised with vectors
containing the DNA fragments that are to be sequenced, enabling the
positioning of various cloned DNA fragments along the chromosome.

2. Gene Libraries:

Construction of gene libraries using restriction endonuclease and then

by cloning the sheared DNA into vectors like cosmid or YAC is
essential to identify the overlapping clones.

3. Screening of Libraries and Constructing Contigs:

STS (sequence targeted sites) or the EST (expressed sequence tags)

helps a lot in sequencing process by facilitating the task of annotation
of the final sequence. When the ordered overlapping clones are
available then the contigs are constructed, which represent stretches
of contiguous sequence ready clones.

4. Sequencing:

After identification of YAC clones these are further fragmented by

restriction endonuclease and again sub-cloned into cosmid. After
identification of overlapping cosmid clones these are then sheared
and cloned in M 13 vectors or pUC plasmid and used for sequencing
using the Sanger method.

5. New Vectors BAC & PAC and the Shot Gun Approach:

YAC clones are found to be unstable and chimeric in nature, so the use
of alternative vectors such as BAC (Bacterial Artificial Chromosome)
or the PAC (PI derived Artificial Chromosome) became necessary,
within which-100 kb can be cloned easily.

With the aid of computers and special software ends the every BAC are
sequenced and then matched with other BAC clones and overlapping
are detected. This process reduces the number of BAC clones to be
sequenced fully and it is more powerful technique.

The genome sequencing in Arabidopsis thaliana was followed by the

efforts of sequencing the genomes in several crop plants like cereals,
oilseeds, legumes, vegetables, etc. The plant genomes that are
sequenced or targeted to be sequenced during the first decade of the
present century have been listed in Table 22.2.

Arabidopsis Genome Project:

Three American groups namely Meyerowitz, Somerville and Goodman

at three different universities took the first step leading to the
project on sequencing of the Arabidopsis genome later known as AGI.

The first RFLP map was created by Meyerowitz, second by the

Goodman group and later integrated. Initially the conventional clone-
by- clone was dominant approach but later shot gun approach was
followed using cDNA libraries and ESTs.
By 1999 the first report came on chromosome 2 and 4, but now the
whole genome has been sequenced with all the informations.

Few of them are as follows:

1. The genome has approx. length of 145Mbp, of which genes are

present (coding region is 2-2.5 kb) at each 4-5 kb interval.

2. Telomere and centromere regions are full of repeated DNA.

3. Centromeric region has transposons and pseudogenes.

4. Sometimes entire stretches of DNA and genes are duplicated

between chromosomes.

5. Approx. 20% of the genes have signal sequences and target products
into organelles such as chloroplasts or mitochondria. Entire
mitochondrial genome of Arabidopsis is represented on chromosome
no. 4.

6. It has about 20,000-50,000 genes of various functional groups.

Rice Genome Project:

Scientists from Japan started the Rice Genome Programme (RGP) in

1991. In 1998 the second phase of RGP was launched. Now 10
countries are participating in the international Rice Genome
Sequencing Programme (IRGSP).

Monsanto (2000) produced the draft of rice genome of

Japonica variety which has explored the following:

a. The rice genome is estimated to comprise of 420-466Mbp of DNA.

b. Monsanto produced 399 Mb of sequences from 3391 BAC clones in

2000 (not available to public).
c. In 2002 the draft sequence of Indica rice variety was published in
Beijing Genomic Institute and Syngenta published a draft sequence of
Japonica variety.

d. Similar to Arabidopsis, the early work began with random genomic

clones, but in recent years cDNAs and ESTs have supplied a large
number of markers. IRGSP has made the progress in preparing contig
maps employing YAC and BAC.

e. From the sequence data available, it has been reported that the total
number of genes in rice is not much larger than in Arabidopsis and
most of the genes of two plants show homology.

f. According to available data on rice from Gen Bank, 28,282,731 bases

of sequences has been submitted from the rice EST sequencing
projects.

g. Average rice gene is 2.2 kbp containing 3.9 exons and 2.9 introns.
The density of rice gene is one gene per 5.7 kbp.