Focus on: Molecular Markers
SUBMERGED RICE
Using Genetics to Streamline Artificial Selection
Introduction
Since its earliest cultivation about 10,000 years ago, the history of rice has been intertwined with that of humans. Today, rice is a staple food in the diets of billions of people around the world. However, as climate changes, so do farming conditions. Many rice species can survive a short time under water but when big, heavy rains come and rice fields are flooded for more than a week, the plants die.
Rice can survive underwater, but not for long.
A two week flood will kill nearly all rice plants.
In low-lying areas in India and Southeast Asia, as local weather patterns change due to global climate change, fields that were once productive rice farms are in jeopardy from prolonged flooding events. Look deeper to explore how rice feeds people worldwide and see how genetic modification via sophisticated artificial selection can help rice "hold its breath" underwater when the floodwaters come.
How can we protect rice against floods?
About the Organism
Rice is a type of grass. Like other grasses, the root system secures the plant in the ground. A mature rice plant has a main stem, from which the long flat leaves grow. Side stems, called tillers, surround the main stem. Each tiller has the potential to produce a panicle, or flower head, in addition to the main stem.

Closed flowers, which contain the plant's reproductive organs, develop on the panicle. After pollen is transferred from the anther onto the stigma, it travels down the style to the ovary where the ova are fertilized. A seed begins to form. Each flower develops into a single kernel of grain. Because the pollination occurs within a closed flower, rice rarely receives pollen from other plants; instead, it pollinates itself—a form of inbreeding called self-pollination.

The edible part of the rice plant is the seed.
Plant Reproduction
The female reproductive parts are the pistil, with its sticky stigma opening into the tubular style that connects to the ovary. The male reproductive structure is the stamen, comprised of the thin filament on top of which sits the anther, which contains lots of pollen.
If you've ever eaten rice, you have eaten those grains, the plant's seeds, which contain the endosperm surrounded by a bran layer, a germ layer, and the outer, inedible husk or hull. After the husk is removed, the kernel that remains still has the endosperm, germ and bran layers; that is brown rice. Further processing, called milling, removes the germ and bran layers so that only the endosperm remains; that is white rice.

The seed is like a lifeboat, packaged up to support the next generation. It contains the new plant, the embryo, and the endosperm contains resource to support it.
The Endosperm
The endosperm is the tissue in a seed that acts as food storage for the developing embryo. The endosperm usually contains starch with some protein and other nutrients.
Germ Layer
The germ layer is the embryo, the part that will grow up to be a new plant.
Think and Apply
If rice plants are planted too close together, as they grow, some of the tillers can get too shaded by nearby plants and die back. Why would that be a problem for rice farmers? What planting changes would you recommend for future fields?
Answer:
Tillers are the primary source of rice seed because they produce the majority of panicles, or flower heads. Farmers want to maximize the number of seeds they produce, so they should plant seedlings with a little more space in between each plant, to give as many tillers as possible enough sun to grow and produce panicles.
Rice is grown worldwide. Increasing floods in the rainfed lowlands (shown in green) threaten rice production in some areas.
Rice grows in many different environments worldwide, from deserts to terraced hills to wet low-lying areas. According to the Food and Agriculture Organization of the United Nations, the total commercial harvest of rice in 2014 was 741 million metric tons, harvested from nearly 163 million hectares. Most of the rice was produced in Asia, with China, India, Indonesia, Bangladesh, Vietnam, Thailand, Myanmar, and the Philippines producing the most. In the United States, which ranked 11thin the world for rice production in 2014 (10 million metric tons), rice is grown in Arkansas, California, Louisiana, Mississippi, Missouri, and Texas.

Although rice is grown in many different environments, 75% of rice is produced in irrigated lowland environments. The muddy conditions inhibit competing weed species, giving the rice a chance to grow well and produce a high yield of grains.
How does rice grow?

Rice comes in a rainbow of different
colors, shapes, sizes and tastes.
What kind of rice have you eaten? White rice? Brown rice? Basmati rice? Rice, Oryza sativa, is a cultivated, inbreeding species that was domesticated in Asia from an ancestral species. Today, thousands of varieties of cultivated rice (Oryza sativa) exist and contain a wealth of genetic diversity.

Different varieties of rice have different traits including taste, colors, firmness, and texture. For example, basmati rice has a long slender grain with a fragrant aroma. In contrast, the varieties used as sushi rice are short-grained and high in starch needed to create the stickiness required for sushi dishes. Rice plants also differ in their production characteristics. Some are tall, others short, some can survive colder temperatures, or salty soil, or extended periods of time in wet ground. The wealth of genetic variation within the different varieties of Oryza sativa and in closely related species offers researchers different traits and genetic material to create varieties that can be successfully farmed in a wide range of environments.
How Nutritious?
Rice, a staple food for billions of people worldwide, is a healthy, low fat, low cholesterol food. It's a great source of complex carbohydrates while it's relatively low in calories and naturally low in sodium.
The polishing process that transforms brown rice into white rice removes bran and germ layers leaving only the endosperm behind.
Think and Apply
Look closely at the nutrition labels above and you'll see that brown rice has a tiny bit more fat and dietary fiber than white rice. Why?
Answer:
Brown rice is a whole grain; it contains the endosperm, the bran layer and the germ layer. Those outer layers contain the little bit of healthy fat and the extra dietary fiber. White rice has only the starchy endosperm layer.
As you see in the labels above, brown rice and white rice have similar macronutrient content. However, that's not the whole story. Human bodies also need micronutrients, elements or substances, like minerals and vitamins, which are crucial in tiny amounts for normal growth and metabolism. Various micronutrients have different roles: some transport substances, some are important for vision, growth, or fighting infections. Others help make proteins and DNA, while others help the blood and other body tissues function correctly.

Carefully examine the table below, and you'll see that brown rice provides higher amounts of many vitamins and minerals, including thiamin, niacin, vitamin B6, magnesium, phosphorus, copper, manganese, and selenium. In fact, micronutrient deficiency is a problem in areas where un-enriched white rice makes up the bulk of people's diets.
Macronutrient
Macronutrients are the carbohydrates, fats, and proteins. The human body needs many grams of each macronutrient each day. Macronutrients get digested (broken down), assimilated, and used by the cells in our bodies.
Think and Apply
Enrichment is a process of adding back in nutrients that have been removed or supplying extra levels of nutrients in a low-nutrient food. How do the micronutrients supplied by enriched white rice compare to those supplied by brown rice?
Answer:
Enriched white rice has added thiamin and niacin; levels are similar to brown rice. The amount of folate and iron in enriched white rice are much higher than the amount in brown rice
Why don't people simply eat more brown rice for more nutrients? One reason is that brown rice is harder to store than white rice. When stored in warm or moderate temperatures, the oils in the bran layer goes rancid, spoiling the rice. Brown rice also takes longer to cook and thus requires more fuel for cooking.
The Challenge:
Surviving Submergence

Rice fields in many areas around the world are prone to damaging flooding. Global climate change increases the risk of flooding.
Rice is a staple food for half of our world's population. In some places, more than half of the calories a person eats come from rice farmed by their families. Millions of farmers' often the poorest farmers in the world, lose their rice crops when heavy rains come and the rice plants are flooded. Local weather patterns have been changing due to global climate change and farmers are looking for varieties of rice that can survive flooding.
Though all plants need water to grow, most plants can't be too wet for too long. Rice plants can tolerate wetter conditions than most; they can grow in very wet soils and can even tolerate short periods underwater. Farmers often take advantage of rice's ability to tolerate water and briefly flood fields to kill weeds. However, flooding that lasts more than a few days can destroy a rice crop. When a rice plant is underwater, it doesn't get much sunlight or the carbon dioxide it needs for photosynthesis. With too much time underwater, the photosynthetic pigment chlorophyll degrades. The plant's ability to utilize energy is constrained. The plants use their energy reserves trying to grow quickly to get above the water, but for most rice plants, that extra expenditure of energy results in the plant's death after three days of flooding.

Some varieties of rice have traits that allow them to tolerate more water than others. Several varieties have a "snorkel" gene, which allows a rice plant to grow quickly to escape floodwaters. The snorkel trait is useful in some areas, but only if the field stays flooded the entire growing season. The long, thin plants can't hold themselves up once the floodwaters retreat.

A different trait, sometimes called "scuba", gives the plant the ability to "hold its breath" underwater by going dormant when submerged. For many years, plant breeders have known of a few rice varieties that could survive 14 days underwater. These plants, however, were traditional varieties that produced very few grains of rice per plant and had low yield. Over many years, plant breeders tried to improve the undesirable characteristics of the "scuba" varieties but were unsuccessful; according to rice breeder David McKill, "we didn't understand the genetics."

The challenge facing rice breeders was clear: help poor farmers cope with increased flooding by creating a rice variety that tolerates many days underwater AND produces a high grain yield. Meeting this challenge would require finding the submergence tolerance or "scuba" gene, and then using genetic knowledge and traditional plant breeding to move the "scuba" gene into rice varieties with desirable traits.
The Solution
This solution starts with a phenotype—a plant that can survive two weeks underwater, the scuba trait. Creating a high-yielding scuba variety occurred in three steps. First, researchers needed to determine the genetic region and then the gene responsible for the scuba, or submergence-tolerance, trait in the traditional variety of rice. Second, the scuba gene had to be moved into a high-yielding variety of rice by making controlled crosses and by using used genetic markers to accelerate traditional plant breeding techniques. And finally, the high-yielding scuba variety had to be field-tested by farmers to see how it fared in real-world floods.

Finding the scuba gene started with a cross between a desirable variety (P1) and the donor variety (P2) with the scuba gene. Researchers then screened the segregating (F2) generation. They looked for which regions of the chromosomes associated with the ability to survive under water. They found the Sub1 region on chromosome 9, which contains 13 genes.
>Part 1: Finding the "Scuba" Gene

Finding a gene is a lot like looking for a needle in a haystack. Even rice, which has a small genome of only 430 million base pairs, has at least 30,000 genes. Researchers can't search them individually. In the hunt for genes, scientists rely on a traditional tool: a cross. To find the scuba gene, researchers began with a traditional, poor yielding rice variety capable of surviving 14 days underwater. They crossed it to a well-known, higher-yielding rice variety. Then they screened the offspring from the cross and looked at the genes.

The trait for submergence tolerance lies in a region on chromosome 9 that the geneticists called Sub1 for submergence. The region, also called a quantitative trait locus or QTL, contains 13 genes. Scientists knew that genes in this area were involved in producing ethylene—a plant hormone. Researchers worked to zoom in on the specific gene and discovered that only one of the area's genes, sub1A, has a version that occurs only in plants that can survive underwater for long periods. It differs from the "normal" version of the gene by only one DNA base. The DNA substitution changes an amino acid in the resulting protein, which helps plants survive underwater without oxygen.
Quantitative Trait Locus (QTL)
A quantitative trait locus, or QTL, is a gene or chromosomal region that affects a quantitative trait like yield, height or the ability to survive underwater. Many genes influence quantitative traits.
>Part 2: Moving the gene into high yielding varieties

Previous attempts to move the scuba gene into a high-yield variety of rice using traditional tools of crossing and back-crossing had failed because too much unwanted genetic material was traveling along with the scuba gene. However, once researchers knew the location of the scuba gene, they could use a powerful new set of tools - molecular markers - to target and speed the traditional plant breeding process.

A molecular marker is essentially like a genetic bookmark—an identifiable sequence of genetic material. Plant breeders and geneticists used molecular markers in two different ways to make high yield scuba rice. First, they used markers to screen whether or not plants had the scuba gene. This screening allowed geneticists to use the plant's genetic material, or genotype, instead of having to grow plants, submerge them, and evaluate their phenotype. Plant breeders then used more markers to eliminate unwanted genetic material traveling with the sub1a allele. The following video explains in more detail how molecular markers work.
Marker-Assisted Selection: Using genetics to streamline artificial selection
The development of scuba rice used both modern and traditional tools. At its core, the process relied on a traditional cross to move the scuba gene from an otherwise undesirable donor variety. Then plant breeders used another traditional tool, back-crossing to the desirable variety, to minimize undesirable genes dragged along with the scuba gene. The modern genetic tools saved plant breeders time by screening plant DNA directly after the initial cross. Marker-assisted selection combined with the back-crossing techniques, allowed them to eliminate unwanted genetic material with a degree of precision impossible with traditional methods.
>Part 3: Field testing
When scientists moved the scuba gene into the popular Indian variety Swarna, they achieved success: the plants were yielded a lot of grains and could survive underwater for many days. Later they moved the scuba gene into other popular varieties like Samba Mahsuri, IR64 and others, through the same process. But those successes are not the end of the story.

The varieties need to be tested in the field to make sure that they work well under field conditions. Researchers conducted many tests, comparing the varieties with and without the sub1 gene. For example, they looked at how well the plants survived flooding (Panel A) and the yield (Panel B).
Percentage of plants with and without the sub1 gene that survived 10 days of flooding plus 10 days to recover. Seedlings were 21 days old at the beginning of the experiment. Data are means of four replicate treatments. Vertical bars represent 1 standard deviation of the mean. Grain yield of plants with and without the (ital) sub1 gene after a growing season without floods. Data are means of four replicate treatments. Vertical bars represent 1 standard deviation of the mean.
Panel A (left) shows the percentage of plants with and without the sub1 gene that survived 10 days of flooding plus 10 days to recover. Panel B (right) shows the grain yield of plants with and without the sub1 gene after a growing season without floods. Hover over each panel for more details.
Time-lapse video shows a field trial of flood-tolerant scuba rice.
Think and Apply
Based on the video and the data provided, what was the impact of adding the Sub1 gene to the IR64 variety (video) and Swarna variety (Panels A and B)?
Answer:
The impact was powerful! Varieties with Sub1 survive flooding much better. The yield of the Sub1 varieties is no different than the yield of the originals in seasons without flooding.
Of course, the real measure of success is rice farmers' harvests. How have the Sub1 varieties been used by farmers in flood prone areas and how have their harvests changed?
Nakanti Subbarao, a farmer from Andhra Pradesh in India, was one of the first in his community to adopt a scuba variety (Swarna-Sub1). After a 3-week long flood, he recovered 70% of this rice variety and much less of the other varieties he planted. He shared Swarna-Sub1 seeds with nearby farmers, which led to 800 hectares of the variety grown in his village, and nearby areas the next year.
Sitaram Yadav from Uttar Pradesh, India, tried three scuba varieties (Swarna-Sub1, IR64-Sub1, Samba-Mahsuri-Sub1). His farm was flooded three times that season. As he was about to harvest a decent yield when most of his fellow farmers had terrible harvests, he called the new varieties, "nothing less than a miracle."
Virender Thakur from the Mahottari District in Nepal tried Swarna-Sub1 in a field that flooded often. His field was submerged for more than two weeks, but he still obtained a decent yield. His neighbors, who grew other rice varieties, lost their entire harvest. He believed that Swarna-Sub1 has less disease infection than Swarna and that the scuba rice also tastes good. The following year, he shared his seeds with nearby farmers.
Conclusion
Farmers need to produce more food from land with environmental challenges as global climate changes, flooding becomes more prevalent, and the population continues to increase. In the case of scuba rice, plant breeders used a combination of traditional and modern tools to create a rice variety that can survive weeks of flooding. Once plant breeders knew the location of the scuba gene, they were able to use molecular markers to guide and speed traditional crosses and back-crosses to create a variety that includes the scuba (Sub1) gene and excludes unwanted genes from the donor plant. The additional precision plant breeders could achieve using genetic tools allowed for success where purely traditional approaches had failed. By using modern genetic tools together with traditional selection techniques, plant breeders can develop new varieties more precisely and rapidly.

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