Exploring the Genotype of the Tea Plant (SABINA)

Raising and exporting black tea is an activity of major importance in Africa today; Africa  has become the world’s second largest tea exporting region after India. Tea has been grown in China for perhaps a millennium “by hand,” with farmers seeking out and hand-selecting the tea bushes producing the best yield and quality of tea. Since tea growing reached the West in the 19th century, however, tea cultivation has rapidly incorporated techniques of genetics and, most recently, of molecular biology. While tea breeders continue to walk the fields daily to monitor the health of crops and to seek out the most promising bushes, the real action today is in the laboratory, where the breeder’s skill is supplemented by work done at the molecular scale with instruments of enormous complexity and cost.

Pelly Malebe, a doctoral student in the SABINA network, has been swept up in this genetic revolution and is eagerly helping to help push it along. She works under the supervision of Prof. Zeno Apostolides at the University of Pretoria’s Department of Biochemistry, riding the crest of an agro-technological revolution. The instruments of the present can not only analyze and compare the genes of the tea plants, but also allow biochemists to accomplish research feats undreamed of just a decade ago.

Pelly Malebe

Pelly is a member of a research partnership between the University of Pretoria and Malawi, where the Tea Research Foundation of Central Africa (TRFCA) is located. Another of her SABINA colleagues is Nicholas Mphangwe, a Malawian who is actively involved in the TRFCA’s breeding program. Their work carries considerable weight in Malawi, where the tea crop provides about nine percent of the country’s foreign exchange and about five percent of the world’s output.

The primary focus of Pelly’s work is to locate and understand the sections of tea DNA that help the plant resist drought. Such a section is known as a marker, a gene or DNA sequence with a known location on a chromosome that can be used to identify the trait of interest in an individual or species. This is critical for tea, which prefers at least 50 inches of rain a year, and begins to drop its leaves when rainfall is not sufficient. Because Pelly is a native of the dry Limpopo province of South Africa, she is no stranger to drought, and her experience with dried-up crops and hungry farmers lends urgency to her work.

As a master’s student at the University of Pretoria in 2009, Prof. Apostolides assigned her the task of searching for a genetic marker in tea plants that are relatively drought-hardy. She proved adept at picking up the complex techniques of tea genetics and was soon able to identify a putative marker for cultivars that show high resistance to drought. A cultivar is a plant selected by growers for certain traits and then vegetatively propagated by stem-cuttings so the next generation will have an identical genome. Virtually all food crops and ornamental plants sold today are cultivars that have been selected for certain traits; very few wild plants are used for commercial purposes.

Such activities are by no means unique to tea; the analysis of genetic variation is an essential part of most plant genetics and crop improvement programs. Knowledge of DNA sequences has become indispensable for basic biological research and in numerous applied fields such as diagnostics, biotechnology, and forensic biology. The type of analysis depends on understanding the plant’s DNA and determining the precise order of the four bases (adenine, thymine, cytosine, and guanine) that function as the “letters” of the DNA alphabet (arranged in base pairs because of the double helix shape of DNA). The “words” that are formed by various arrangements of the letters determine the output, traits, and reproduction of the genome – for tea and every other organism, from virus to human. Any change in the spelling of these words is critical to DNA analysis as it might signify, for example, greater (or lesser) resistance to drought.

Pelly spends much of her time analyzing the DNA sequences that determine the genetic makeup, or genome, of tea. The huge number of base pairs in the genome makes this analysis extremely complex. The tea genome, for example, is estimated to be about four billion base pairs long – even more than the 3.2 billion base pairs of the human genome. Such a “library” of genetic information is so daunting that it has taken many decades to decipher it. The structure of DNA was established as a double helix by James Watson and Francis Crick in 1953, but not until the early 1970s could scientists reliably interpret the sequence of even a few DNA fragments in the laboratory.

Since the development of automated analysis, however, DNA sequencing has advanced rapidly. When Pelly first began to learn genotyping several years ago, she used a relatively slow technique known as RAPD PCR, or random amplified polymorphic DNA polymerase chain reaction. This technique limited her analyses as it is time consuming and has a low reproducibility rate. In the brief time since starting her PhD research in August 2011, she has moved to a more advanced genotyping technique that is orders of magnitude faster.

“This technology is evolving so rapidly that there seems to be a new instrument every day,” she said.

One reason Pelly and her colleagues depend on current generation technology is that the sequences of DNA that indicate various behaviors in tea involve many genes. She has found, for example, that about 40 percent of tea strains, or cultivars, which she has worked with have sequences of DNA that indicate some degree of drought tolerance. But for best results she needs to know exactly which base-pair combinations are generating the best drought tolerance. She also needs to recognize as many DNA markers as possible. She identified a 1,400-base-pair marker during her MSc work, but current technology will bring her far more information.

Pelly and Nick are also looking for several kinds of selection methods for different traits, including the ability for better yield, disease resistance, and cold hardiness. From the breeding work done using conventional means over many years at the TRFCA, many desirable cultivars are already known.

The trait of drought resistance is a particular concern for Africa, and many growers are concerned by the prospect of climate change. To refine her early work on drought resistance, Pelly relies on cultivars gathered by Nick and others from the Malawi fields, or from elite mother bushes gathered there in previous years. When a promising cultivar is recognized, it may be propagated at the TRFCA plant nursery. The DNA itself is extracted from a tea leaf with a special kit. Then the DNA is treated with anti-DNAase enzyme to prevent other enzymes from breaking up the DNA, and protease enzymes are added to clear away unneeded protein, producing a pure sample of DNA.

“Then we run it through analysis looking for markers at the most likely DNA sequences,” said Pelly. “This technique is random, because we want to look everywhere. When we find possible sequences, then it’s up to me to identify which are most likely associated with the trait.”

Once a cultivar that is thought to have genes involved in drought resistance has been identified, the next step is to test the plants in the field. Instead of waiting for a drought, however, this can be now done in real time. Thanks to a European Union grant to POL-SABINA (SABINA’s sister organization at the University of Pretoria) a rain shelter has now been erected at the TRFCA in Malawi. The tea is planted beneath the shelter, which can then simulate varying degrees of drought for the plants.

“This will show whether the markers are there just by chance or whether they signal drought tolerance,” she said. “Then if you find the same marker in a second plant that is known to be drought-tolerant, you have a good clue. We got a head start using a small number of drought-tolerant plants identified in a previous experiment which are already available and perfectly valid for our testing. Eventually we hope to expand the numbers. This helps us with the breeding process – having a known trait and being able to use the plants with the trait as parents for the next generation.”

Prof. Apostolides has been working with Kenyan and Chinese colleagues to plan cooperative sequencing research around the world that will be placed in the public domain. By promoting the sharing of knowledge with other countries, SABINA researchers are honoring the traditional scientific practice of openness.

Story by Alan Anderson  SIG, 11 February 2013