GENET archive


2-Plants: Marker assisted selection leeds to improved plant varieties

                                 PART I
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TITLE:  Marker Assisted Selective Breeding
SOURCE: The Institute of Science in Society, UK, Press Release
DATE:   8 Sep 2005

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Marker Assisted Selective Breeding

Prof. Joe Cummins gives the current state of play in how molecular
genetic analysis can aid in selective breeding without genetic modification

The fully referenced version of this article is posted on ISIS members'
website Details here http://

Quantitative traits not determined by single genes Genetically modified
(GM) crops are based on inserting synthetic foreign genes mainly from
bacteria, to impart herbicide tolerance or insect resistance into the
genomes of crop plants. This technology has so far provided little if any
increase in yield, stress tolerance or long-term resistance to microbes
or nematodes. Traditional breeding of crops and animals has been based on
the use of genetic markers that are inherited. The main agricultural
traits governing yield (or size), stress resistance or long-term disease
protection are quantitative trait loci (QTL, 'loci' is another word for
genes). One of the founders of the study of population genetics, Ronald
A. Fisher, described QTL as many independent loci that added together to
determine traits such as size [1]. QTL are seldom tightly linked on a
chromosome and the loci are dispersed over many chromosomes in the
genome. Selection of QTL traits has been inherently slow and meticulous,
but has resulted in major improvements to crops and livestock.

While Fisher believed that QTL were made up of very many genes each
adding small increments to a trait, recent findings indicate that some
QTL may be made up of a relatively small number, say twenty or so,
genetic markers that could be easily selectedprovided they could be
identified. Currently, it appears that many QTL may have relatively few
loci but some important QTL may be closer to the very large number of
genes envisioned by Fisher, in which case, identifying and selecting such
traits by the molecular markers are unlikely to be cost-effective.

Molecular markers can be used to aid selective breeding There is a
growing arsenal of molecular markers (polymorphisms) that aid in
identifying QTL and selecting them for crop and animal enhancement. The
process ofusing such markers is called marker-assisted selection (MAS),
which differs from genetic modification because the genes being selected
for crop or animal improvement are not altered in any way. The molecular
markers used in selection are probed using sequences from a gene bank and
identified. The markers used to probe the progeny of a cross are not the
QTL genes themselves but they are close to the QTL on the genetic map. Of
course the markers can be used to determine the molecular identity of the
QTL, but the molecular marker is used even when the QTL is identified
because the marker is cheaper and quicker to use to identify a large
number of progeny. Recombination may separate the marker from a QTL, but
the closer the marker is to the QTL, the more remote is the chance of
separation by recombination. The more polymorphic markers available for a
breeding programme, the more effective it will be.

There are several types of mk but requires a relatively large amount of
DNA and is rather expensive in a large screening program [2]. RAPD
utilizes low stringency polymerase chain reaction (PCR) amplification
with single primers of arbitrary sequence to generate strain-specific
arrays of anonymous DNA fragments [3]. The method requiresolecular
markers used in MAS; these include restriction fragment length
polymorphism (RFLP), random amplification of polymorphic DNA (RAPD),
amplified restriction fragment length polymorphism (AFLP), single
sequence repeats (SSR) and single nucleotide polymorphisms SNPs [2]. RFLP
involves the use of restriction enzymes to cut chromosomal DNA at
specific short restriction sites, polymorphisms result from duplications
or deletions between the sites or mutations at the restriction sites.
RFLP provided the basis for most early wor tiny DNA samples and analyses
a large number of polymorphic loci [2]. AFLP requires digestion of
cellular DNA with a restriction enzyme, then using PCR and selective
nucleotides in the primers to amplify specific fragments [4]. The method
measures up to 100 polymorphic loci and requires a relatively small DNA
sample for each test [4]. SSR analysis is based on DNA micro-satellites
(short-repeat) sequences that are widely dispersed throughout the genome
of eukaryotes, which are selectively amplified to detect variations in
simple sequence repeat [5]. SSR analysis requires tiny DNA samples, and
has a low cost per analysis [2]. SNPs are detected using PCR extension
assays that efficiently pick up point mutations [6]. The procedure
requires little DNA per sample and costs little per sample once the
method is established [2]. One or two methods are used in a typical MAS
breeding programme.

MAS has been employed in breeding cereals, and extensively so in maize
breeding. Corporations including Monsanto and Syngenta have invested
heavily in the programme. SNP appear to be the dominant marker for
selection. Wheat has seen less progress in MAS than maize, but there is
good success in the area of quantitative disease resistance. Rice has
also seen extensive activity in MAS centering on pyramiding disease
resistance genes. SNPs appear to be identified for all the major cereals
[7]. MAS is being used to improve forage crops through QTL for nitrogen
use efficiency, and there was a strong response [8]. The pome fruits,
apple and pear, have extensive MAS programmes, mainly based on RFLP,
RAPD, SSR and AFLP. The traits being selected include fruit production,
storage and disease resistance [9]. A global strategy using MAS for
livestock genetic improvement in the developing world was proposed. QTL
mapping would be used in genetic improvement and to bring together
desirable traits from around the world [10]. It has been proposed that
assessment of genetic markers will greatly enhance the conservation of
genetic diversity in wild crop relatives [11], and the information from
wild crop relatives could be directly employed in MAS of the crop plant.

Does MAS actually work? A recent review by William Hill of Edinburgh
University focused on the QTLs for oil production in maize and for body
size in chickens. In neither case could individual QTL with substantive
quality be detected. Instead, identified QTLs created small additive
increments that could be selected, but only with patience [12]. Hill's
report suggested that Fisher's view of QTLs prevailed and that the use of
MAS might not be cost-effective. It may be that MAS is effective in
traits such as disease resistance and certain agronomic performance but
that important traits such as oil production in maize or body size in
chickens are most effectively bred using traditional selection methods.

Farmers in developing countries and even some farmers in the developed
world face the growing control of seed production by a few multinational
corporations. One solution has been to help the farmer breed varieties
tuned to the local environment and free of the greedy demands of seed
corporations. It is highly unlikely that indigenous farmers will take to
MAS and molecular genomics. However, those scientists working with
indigenous farmers would recognize markers linked to valuable agronomic
traits and pass on that knowledge to the indigenous plant breeders to
assist them in making selections that are beneficial.

In the long run it seems likely that MAS will play an important role in
plant breeding, even though it may not be as large as has been claimed by
advocates. MAS should not affect organic certification because transgenes
are not introduced into the crop. Molecular genetics is used only in
analyzing the crosses. Nevertheless, MAS has far more to offer in crop
and animal improvement than genetic modification.

                                 PART II
------------------------------- GENET-news -------------------------------

SOURCE: ISB News Report, USA, by Motoyuki Ashikari and Makoto Matsuoka
        file attached: sep0504-1.jpg
DATE:   Sep 2005

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QTL cloning for grain productivity and plant height

Rice (Oryza sativa L.) is a staple food and approximately 50% of the
human population depends on rice as their main source of nutrition. In
particular, it is the most important crop for people living in the
monsoonal areas of Asia where rice has a long history of cultivation; it
is deeply ingrained in the daily lives of Asian people.

Rice is a model monocot because it has the smallest genome size (390 Mb)
among the major cereals, its genome is syntenic with the genomes of other
cereals, and it can be transformed easily. As a result, The International
Rice Genome Sequencing Project (IRGSP) was launched in 1998 to sequence
the rice genome; the task was completed in 2004. These accomplishments
and recent technological innovations have greatly facilitated gene
cloning and provided a new breeding strategy for rice as well as for
other major cereals.

In contrast to monogenic characteristics, such as disease and insect
resistance, many important agronomic traits including yield, heading
date, culm length, grain quality, and stress tolerance show continuous
phenotypic variation. These complex traits usually are governed by a
number of genes known as quantitative trait loci (QTLs) derived from
natural variations. Although these polygenic characteristics, including
QTLs, were previously very difficult to analyze using traditional plant
breeding methods, recent progress in rice genomics has made it possible
to search QTLs.

QTL analysis is a powerful approach to discover agronomically useful
genes. Grain number and plant height are important traits that directly
contribute to grain productivity. Why is plant height important for grain
production? Dwarf rice and wheat varieties were developed by classical
plant breeding methods, contributing to the green revolution in the
1960's. Higher yields were obtained from these dwarf crops because their
short stature reduced lodging from wind or rain1,2. Our lab has aimed to
identify genes of QTLs for grain number, Gn1, and plant height, Ph1, not
only to elucidate molecular mechanisms for grain productivity, but also
to utilize these genes for breeding3.

A choice of parental lines that show wide phenotypic variation in the
targeted traits is necessary for QTL analysis because QTL detection is
based on natural allelic differences between parental lines. An indica
rice variety, Habataki,

and a japonica variety, Koshihikari, were chosen in QTL analysis since
not only do they exhibit large differences in grain number and plant
height, but also many molecular markers are available. QTL analysis using
progenies from the cross with Habataki and Koshihikari revealed the
presence of five QTLs for increasing grain number (Gn1-5) and four QTLs
for plant height (Ph1-4). The most effective QTLs for grain number, Gn1,
and plant height, Ph1, were chosen as targets for cloning.

QTL cloning is facilitated by using nearly isogenic lines (NILs) carrying
only one target QTL, because NIL can eliminate the effects of other QTLs.
By using a suitable NIL, the QTL of interest in the NIL can be treated as
a single Mendelian factor. We produced the NIL-Gn1 and NIL-Ph1 lines
carrying the Gn1 or Ph1 region from Habataki in the Koshihikari
background and used these lines for cloning. Base on fine mapping
analysis of QTL-Gn1, we found that Gn1 could be divided as two linked
QTLs (QTL-Gn1a and QTL-Gn1b). We focused on the Gn1a, since it could be
mapped between two molecular markers.

Positional cloning and molecular characterization revealed that Gn1a
encodes a cytokinin oxidase/dehydrogenase, OsCKX2, an enzyme that
degrades a phytohormone cytokinin. The OsCKX2 gene of Koshihikari and
Habataki consists of four exons and three introns and encodes proteins of
565 or 563 amino acids, respectively. A comparison of the DNA sequences
between the cultivars revealed several nucleotide changes, including a
16-bp deletion in the 5'-untranslated region, a 6-bp deletion in the
first exon, and three nucleotide changes, resulting in amino acid
variation in the first and fourth exon of the Habataki allele. Both
OsCKX2 alleles of Habataki and Koshihikari encode an enzyme capable of
degrading cytokinin. However, the expression level of OsCKX2 in Habataki
was lower than in Koshihikari, resulting in less cytokinin accumulation
in the inflorescence meristems of Habataki than Koshihikari. Cytokinin
(CK) is known to influence various aspects of plant growth and
development, including seed germination, apical dominance, leaf
expansion, reproductive development, and delay of senescence. The reduced
expression of OsCKX2 can explain the increased cytokinin accumulation,
and hence, increased grain number. The semi-dominant inheritance of Gn1a
is also consistent with the function of the OsCKX2 enzyme that degrades

On the other hand, Ph1 was located near the sd1 gene that encodes
gibberellin 20-oxidase. Comparing the sequence of sd1 in the Habataki and
Koshihikari alleles revealed that Habataki had a 383-bp deletion in the
SD1 gene, which is the same mutation found in 'IR8', a variety that led
to the green revolution in rice. This observation demonstrated that the
short stature of Habataki mainly depends on the sd1 locus3.

QTL pyramiding breeding

QTL pyramiding is an efficient strategy for crop improvement. This
strategy is based on the combination of desirable QTLs through
conventional crossing using molecular markers. Once desirable QTLs are
detected, a strategy for QTL pyramiding employs the use of NILs harboring
only one target QTL. The NIL-QTLs are produced by backcrossing and marker
selection. A parent line with a positive QTL is backcrossed with a
recurrent parent lacking the QTL. Subsequently, a line that carries only
a positive QTL region from the mother line in the recurrent parent genome
background is selected by molecular markers. The NILs can be used to
accurately evaluate the effect of each QTL individually. Once QTLs with
important effects are identified in this manner, the appropriate NIL-QTLs
are crossed to pyramid two or more QTLs in the same background. The two
NILs, the NIL-Gn1 and NIL-sd1, carrying the Gn1 region or sd1 region from
Habataki in the Koshihikari genome background, were produced by
backcrossing with Koshihikari and using marker selection. We then derived
a NIL-sd1+Gn1 plant with the two desirable QTL alleles derived from the
cross NIL-Gn1 and NIL-sd1. The NIL-sd1+Gn1 has a high yielding, semi-
dwarf phenotype (Fig.1)3.

Future outlook

Food shortage is one of the most serious global problems in this century.
The world population is expanding rapidly due to a significant decline in
mortality rates resulting from advancements in modern medicine and human
health care, while the availability of land for cultivation has
dramatically declined as the result of desertification caused by reckless
deforestation and construction. The global population, now at 6.4
billion, is still growing rapidly and is projected to reach 8.9 billion
people by 2050. Cereals are an important source of calories for humans,
both by direct intake and as the main feed for livestock. Approximately
50% of the calories consumed by the world population originate from three
cereals, rice (23%), wheat (17%), and maize (10%). To meet the expanding
food demands of the rapidly growing world population, crop grain
production will need to increase by 50% by 2025.

Now that genomic information and tools are available for rice, we have to
apply these for human benefits. Today the cloning of genes and production
of transgenic plants are common technologies utilized in plant science.
Such technologies are very powerful and efficient strategies for
producing 'ideal' crop plants. Actually, some transgenic crop plants with
traits such as insect resistance and herbicide tolerance have already
been commercialized. Despite concerns about the impact of GMOs on the
environment and their safety to humans, we think transgenic crops are
necessary to meet the demand for food in the near future. However, we
should not ignore the conventional breeding approach--the QTL pyramiding
approach results from a combination of recent crop genomics and
conventional breeding, and it is efficient for crop breeding. We believe
both strategies, GMO strategies and QTL pyramiding, are necessary and the
cooperation of molecular geneticists and breeders is required to
accomplish this goal.

Fig. 1. Phenotypic characterization of NIL-QTLs. (A) Plant morphologies
and chromosome maps of Koshihikari, NIL-sd1, NIL-Gn1, and NIL-sd1+Gn1.
White and red scale bars indicate 1 m and 20 cm, respectively. (B)
Comparison of plant height, (C) grain number in the main panicle, and (D)
grain number in whole plants for Koshihikari, NIL-sd1, NIL-Gn1, and NIL-
sd1+Gn1. Values in (B) to (D) are means with SD (n = 10 plants). (From:
Ashikari et al. (2005) Science 309, 741-745)


1. Peng et al. (1999) Nature 400, 256-261

2. Sasaki et al. (2002) Nature 426, 701-702

3. Ashikari et al.(2005) Science 309, 741-745

Motoyuki Ashikari and Makoto Matsuoka
Bioscience and Biotechnology Center
Nagoya University, Nagoya 464-8601, Japan

                                  PART III
-------------------------------- GENET-news -------------------------------

TITLE:  GENETICALLY ENGINEERING RICE: New breeds resistant to diseases and
SOURCE: The Nation, Thailand, by Pongpen Sutharoj
DATE:   2 Sep 2005

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This new rice variety does not seem to be a GE rice but produced by marker
assisted selection. I could not find more information about the case.
Hartmut Meyer/GENET

GENETICALLY ENGINEERING RICE: New breeds resistant to diseases and pests

To improve rice quality and yields, the National Centre for Genetic
Engineering and Biotechnology (Biotec) has developed new breeds of
jasmine rice that are tolerant of drought, pests and disease.

The new rice breeds are the result of a research project on rice genomes.
Understanding the rice genome will help scientists to develop new rice
varieties with traits such as higher yield, improved nutrition content,
and better resistance to diseases and pests.

Biotec's director Morakot Tanticharoen said the centre's researchers had
applied information from the International Rice Genome Sequencing
Project, which cracked the genome of the Japanese aromatic rice
Nipponbare, to develop new breeds of Thai rice.

The International Rice Genome Sequencing Project is a collaborative
project among 10 nations to break the genetic code of rice. Thailand is
also one of the participants, along with the United States, Japan,
Canada, Taiwan, South Korea, Britain, France, Brazil and India.

The results of the study have been put in the public domain, so any
country can use them in its own developments.

Morakot said the researchers used the information to further develop
jasmine rice. Even though the rice genome information in the project is
based on Japanese rice, she said the genome information could also be
adapted to other species including jasmine rice, as the DNA structures of
individual rice species do not vary greatly.

The centre has studied sequence data from the project to develop a new
breed of rice that can resist flooding, a major problem for rice farmers
as it causes damage and loss of productivity.

Morakot said the new breed has already been tested in many provinces and
the result was satisfactory. "We found that our new breed can resist
flooding well. It offers higher productivity at 303 kilograms per rai,
compared to the old breed that provides only 50 kilograms per rai," she said.

In addition to flood tolerance, the centre has also developed two other
breeds of jasmine rice.

They can resist bacterial leaf blight and leaf blast disease, which are
major threats.

The director said the two breeds were also being tested. However, to
further improve jasmine rice, the team is now working to combine three
key traits - resistance to drought, bacterial leaf blight, and leaf blast
disease - into one breed so the new breed could tolerate every situation.
The project is likely to move into field trials next year.

From the study of rice genomes, Morakot said the research team could also
understand the DNA sequence of jasmine rice that offered its unique fragrance.

"From this knowledge we can develop a process to turn rice with no
fragrance into fragrant rice," she said.

The centre has submitted a patent registration for the process and it's
now waiting for approval. The centre also plans further study on rice
genomes to give rice special qualities, for example finding genes related
to the quality of rice when cooked in different ways.

This, Morakot said, would bring more added value to Thai rice for export.


European NGO Network on Genetic Engineering

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