Characterization of Perennial Traits in Hybrids Between Maize

Home , Arabis alpina, Perennial grain, Zea diploperennis, Zea perennis

CHARACTERIZATION OF PERENNIAL TRAITS IN HYBRIDS BETWEEN MAIZE

AND PERENNIAL TEOSINTE

CAROLINE COATNEY

(Under the Direction of R. Kelly Dawe)

ABSTRACT

Genetic mapping and quantitative trait locus/loci (QTL) mapping were used to study the genetic factors associated with perenniality in a cross between the annual maize inbred B73 (Zea mays ssp. mays) and a perennial teosinte relative, Zea diploperennis.

The perennial-related traits analyzed were flowering time, tiller number, regrowth after autumn cutback, stay-green, and overwintering. An F2 population of 482 individuals was phenotyped and a subset of 93 F2 individuals was genotyped using 858 single nucleotide polymorphism (SNP) markers generated with genotyping-by-sequencing (GBS). A genetic map was developed that totaled 1,437.5 cM in length with an average of 85.8

SNPs per chromosome. Associations between phenotypes and genotypes were analyzed with composite interval mapping. One QTL was detected, which was for the stay-green phenotype on chromosome 5 that was responsible for 27.0% of the phenotypic variation.

INDEX WORDS: Perenniality, Zea diploperennis, Genetic map, Genotyping-by-

sequencing, Wide hybridization, Stay-green, QTL mapping

CHARACTERIZATION OF PERENNIAL TRAITS IN HYBRIDS BETWEEN MAIZE

AND PERENNIAL TEOSINTE

CAROLINE COATNEY

B.A., Knox College, 2011

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2015

Caroline Coatney

CHARACTERIZATION OF PERENNIAL TRAITS IN HYBRIDS BETWEEN MAIZE

AND PERENNIAL TEOSINTE

CAROLINE COATNEY

Major Professor: R. Kelly Dawe Committee: Katrien Devos Wayne Parrott

Electronic Version Approved:

Suzanne Barbour Dean of the Graduate School The University of Georgia August 2015

ACKNOWLEDGEMENTS

I would like to thank my committee members, Dr. Katrien Devos and Dr. Wayne

Parrott, for not only giving me guidance and advice on my research, but also for supporting me in my interest in science communication. Both have gone out of their way to make sure certain opportunities crossed my path. I would also like to thank my adviser,

Dr. Kelly Dawe, for introducing me to the world of maize research and teaching me the rich history of maize genetics. The Zea genus, especially the teosintes, has become special to me in a way I thought it never would.

Finally, I would like to acknowledge my husband, Peter. He has been wonderful beyond measure during my time in graduate school. From leaving hidden stashes of chocolate around the house for me to find to helping me with unpleasant field work on his time off, he has buoyed my spirit when it has needed it most. My success is a direct result of his support.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... iv

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ...... 1

2 MATERIALS AND METHODS ...... 10

3 RESULTS ...... 19

4 DISCUSSION AND CONCLUSIONS ...... 34

REFERENCES ...... 42

LIST OF TABLES

Page

Table 1: Means and standard deviations (s.d.) for phosphorous (P), potassium (K),

calcium (Ca), magnesium (Mg), zinc (Zn), manganese (Mn), and pH levels for six

soil samples from field ...... 12

Table 2: Labels and phenotypic groups of plants genotyped-by-sequence ...... 13

Table 3: Means, standard deviations (s.d.), and confidence intervals for flowering and

tiller traits for 482 F2 plants along with observed values for the eight Z.

diploperennis individuals and maize B73 parent ...... 21

Table 4: Fall regrowth and overwintering scores for parents, 482 F2 plants, and Z.

diploperennis controls ...... 25

Table 5: Number of SNPs, map length, mean distance between SNPs, and density of

SNPs per chromosome for genetic map of F2 hybrids between maize B73 and Z.

diploperennis...... 28

Table 6: Chi-squared significance values for segregation patterns of SNPs across all

chromosomes ...... 29

Table 7: Chromosome location and direction of QTL for stay-green phenotype estimated

by composite interval mapping (CIM) in 93 genotyped F2 plants ...... 33

LIST OF FIGURES

Page

Figure 1: Field design for experiment ...... 11

Figure 2: Distribution of flowering dates for 482 F2 plants ...... 20

Figure 3: Distribution of tiller values for 482 F2 plants ...... 22

Figure 4: Distribution of tassel emergence, pollen shedding, silk emergence, and tiller

number values for 93 F2 plants that were genotyped (parents excluded) ...... 23

Figure 5: Regrowth and stay-green phenotypes in F2 plants after cutback...... 24

Figure 6: Maximum and minimum air temperatures from October 2014 to mid-April

2015…...... 25

Figure 7: Genetic map for chromosomes 1-5 for F2 mapping population ...... 30

Figure 8: Genetic map for chromosomes 6-10 for F2 mapping population ...... 31

Figure 9: Genetic map of chromosome 5 plotted with QTL interval and QTL LOD graph

for 93 F2 plants ...... 33

Figure 10: Synteny between maize B73 chromosome 5 (left) and Sorghum bicolor

chromosome 1 (right) at location of stay-green QTL ...... 40

vii

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

Perennial cereal grain crops

The dichotomy between perennials and annuals is one of many used to divide and organize plant species. In recent years, the potential fluidity between perennial and annual states, and the usefulness it could have in an agricultural context, has been seriously discussed in the scientific community (Cox et al. 2002; DeHaan et al. 2005;

Cox et al. 2006; Cox et al. 2010; Glover et al. 2010; Van Tassel & DeHaan 2013;

DeHaan & Van Tassel 2014). Specifically, if the genetic factors differentiating perennial plants from annual plants are discovered, annual crops could be converted to perennials and revolutionize farming in parts of the world.

There are many life history traits that distinguish perennials from annuals.

Annuals propagate by seeds and have one growing season. Perennials multiply and disperse by both seeds and vegetative organs—such as rhizomes, stolons, tubers—and live for two or more growing seasons. These vegetative organs allow perennial plants to overwinter and quickly regrow at the start of the next growing season. Early leaf canopy production can be a weed deterrent since it shades out weedy competitors. Converting annual crops to perennial crops would not only take advantage of early biomass production, but could also bring to fruition several other benefits. Unlike annual crops, perennial crops could reduce soil erosion and store more nutrients and carbon below

ground because of their large root structure and storage organs, and could also require less water and fertilizer since their deeper roots allow access to deeper resources (Grime

1977; Cox et al. 2002; DeHaan et al. 2005). These advantages make perennials particularly well adapted to thrive on marginal lands (Tilman et al. 2009), where they would be most useful to third world subsistence farmers.

The majority of the world’s calories are supplied by cereal grain crops. The major cereal grain crops are rice, wheat, and maize. Since 1998, maize has been the most produced cereal grain crop in the world (FAOSTAT Database, http://faostat.fao.org/).

However, it does not have nearly the same prominence in the realm of perennial research.

Here, we attempt to contribute information and data to elucidate the potential of converting maize to a perennial crop.

The teosinte taxa

Domesticated maize, Zea mays ssp. mays, is one of the most recognizable and most researched plant species in the world. This attention, especially from the scientific community, has revealed the intimate details of its biology as well as the biology of the species most closely related to it, including the teosinte taxa. The teosintes are a group of plants native to a range spanning Mexico, Guatemala, and Nicaragua that is composed of ecologically diverse environments in terms of precipitation, elevation, and temperature

(Hufford et al. 2012). The members of the teosinte taxa include the perennials Zea diploperennis and Zea perennis (the only tetraploid teosinte), and the annuals Zea luxurians, Zea nicaraguensis, and Zea mays. The latter is composed of four subspecies:

(1) ssp. mays, domesticated maize, (2) ssp. mexicana, (3) ssp. huehuetenangensis, and (4) ssp. parviglumis, the progenitor of maize (Matsuoka et al. 2002).

Of these teosintes, Z. perennis and Z. diploperennis are of particular interest to our work due to their perennial habit. Z. perennis, first discovered in 1922 (Hitchcock

1922), was previously known as Euchlaena perennis until it was taxonomically redefined to be included in the Zea genus in 1942 (Reeves & Mangelsdorf 1942). It is an autotetraploid (2n = 4x = 40) found in distinct populations on the mountain slopes of

Jalisco, Mexico (Hufford et al. 2012). Much effort has gone into studying the cytological relationship and hybridization potential between Z. perennis and diploid domesticated maize. Some researchers have focused on the triploid hybrids produced while others have either reduced the Z. perennis genome or doubled the domesticated maize genome before crossing (Emerson and Beadle 1930; Molina 1978a; Molina 1978b; Mazoti and Rimieri

1978; Vina and Ramirez 1994; González et al. 2006; Allen 2005; Takahashi et al. 1999;

Shaver 1964; Shaver 1962; Emerson 1929; Tang et al. 2005; Shaver 1963). Differences found between parental chromosomes include position of centromeres, length of chromosomes, and the presence or absence of knobs.

The second perennial teosinte, Z. diploperennis, is a diploid (2n = 2x = 20).

Originally thought extinct in 1921, Z. diploperennis was rediscovered in 1979 at two locations on the mountain slopes of southern Jalisco, Mexico (Iltis et al. 1979). Iltis et al. describe it as morphologically distinct from Z. perennis by its two different types of rhizomes, less dense root system, increased number and length of tassel branches, wider and longer leaves, and more “robust habit.” Since its discovery, it has become the

preferred perennial teosinte for study most likely due to its ploidy compatibility with domesticated maize and the majority of the teosintes.

Perennial research in maize

Before the rediscovery of Z. diploperennis in 1979, the few attempts made to develop a perennial maize variety were limited to using Z. perennis as the perennial donor. Shaver (1964) made the first attempt to develop perennial maize by trying two different crossing schemes. His first crossing scheme began with crossing Z. perennis with diploid maize and then followed by backcrossing F3 individuals to the maize parent.

This resulted in triploid plants that readily grew shoots and roots from culms separated from the main root mass, which Shaver believed were indicative of perenniality. His second crossing scheme used Z. perennis and colchicine-induced tetraploid maize to produce tetraploid hybrid progeny. After strongly selecting for tillers for several generations, he found 70% of F4 individuals had a significant number of tillers, which he equated with perenniality. However, after backcrossing to tetraploid maize, no plants were perennial. For both crossing schemes, plants were overwintered in the greenhouse.

From his triploid work, Shaver was able to produce a 22-chromosome hybrid that showed what he believed to be the perennial phenotype (Shaver 1964). A low-frequency crossover event that allowed the postulated perennial gene, pe, to be present in combination with the recessive genes for indeterminacy (id) and grassy tillers (gt) in a diploid background was thought to be responsible for the observed perennial phenotype

(Shaver 1967). All material was grown at winter nurseries in either California or Florida.

However, tiller number was used as the primary metric for the perennial phenotype, which some believe to be a potentially misleading technique (Cox et al. 2002).

In the 1980s, the search for the genetic basis of perenniality in Zea shifted away from Z. perennis and towards Z. diploperennis, after the latter’s rediscovery. Mangelsdorf and Dunn (1984) hypothesized in the Maize Genetics Cooperation Newsletter that Z. diploperennis was perennial due to its rhizomes, which they believed was a recessive trait, and that an associated gene was located on the long arm of chromosome 4. Later,

Srinivasan and Brewbaker (1999) crossed Z. diploperennis with eleven Hawaiian tropical maize inbreds. They quantified several morphological traits, including tiller number, in the F1, F2, BC1, and BC2 populations over the course of three seasons in Hawaii.

However, they were not able to produce any perennial plants. Westerbergh and Doebley

(2004) took a quantitative trait locus/loci (QTL) mapping approach and crossed Z. diploperennis with the annual teosinte Z. mays ssp. parviglumis. They scored the F2 population for eight traits associated with branching, withered stems, rhizomes, underground stems and roots, and tillers; however, all plants were dug up for underground characterization at the end of the first growing season, preventing any overwintering analysis. Thirty-eight QTL were found for the eight traits. QTL for six traits mapped near each other on chromosome 2, and QTL for four traits mapped near each other on chromosome 6. Two QTL were identified for rhizomes, which explained less than 12% of the variation. Nine QTL were identified for number of tillers, which explained almost 45% of the variation (Westerbergh & Doebley 2004).

Perennial genetics research in other taxa

Rice

Research on the genetics of perenniality has been most successful with rice, which has led to five perennial rice lines being developed, one of which is in pre-release testing (Batello et al. 2013). None of these lines have been approved for commercial distribution yet. This success is most likely due to the fact that cultivated Asian rice,

Oryza sativa, was domesticated from a perennial species, O. rufipogon. Some cultivated genotypes maintain a weak perennial phenotype with plants producing a ratoon crop under irrigated conditions (Sacks et al. 2003). None survive for multiple years though.

The greatest research strides have come from wide crosses between O. sativa and

O. longistaminata, a wild perennial African species that propagates via rhizomes. Two dominant complementary QTL for rhizomes, Rhz2 and Rhz3, have been mapped to chromosomes 3 and 4, respectively; an additional 15 minor effect QTL for rhizome formation have also been identified (Hu et al. 2003; Hu et al. 2011). Rhz2 corresponds closely to rhizome QTL on linkage group C of Sorghum propinquum, a wild perennial relative of cultivated sorghum (Hu et al. 2003). Rhz3 corresponds closely to a major rhizome QTL on linkage group D of S. propinquum as well as to a dominant rhizome

QTL on linkage group 2a of perennial wildrye (Hu et al. 2003; Yun et al. 2014).

Molecular markers were developed for the rhizome QTL to transfer the traits by multiple backcrosses to the cultivated parent. A perennial cultivar, PR23, was developed that maintained impressive yields over the course of three seasons under tropical lowland paddy conditions. It is currently undergoing pre-release testing in Yunnan Province in

China (Batello et al. 2013).

It is important to note that there are many perennial Oryza species and not all propagate from rhizomes. For example, O. rufipogon, the perennial ancestor of cultivated rice, propagates via stolons, which are horizontal stems that can be above or below ground. However, it is generally thought that rhizomes are more stress tolerant than stolons, thus O. longistaminata was chosen as the perennial parent for breeding. Even so,

Sacks et al. (2006) found that rhizomes and perenniality were not associated in O. sativa/O. longistaminata interspecific genotypes/cultivar full-sib family progeny, with

15% of plants surviving past one growing season without rhizomes. Sacks et al. speculated rhizomes may not be necessary for perenniality, but instead might help plants manage and endure abiotic stresses.

Sorghum

Rhizome, tiller, and regrowth QTL have been identified and mapped in an F2 population from a cross between the cultivated annual Sorghum bicolor and Sorghum propinquum, a closely related wild perennial species (Paterson et al. 1995). Winter survival and regrowth was measured in the field in Texas; 92.2% of F2 plants survived and 46.3% of BC1 plants survived. Paterson et al. (1995) found that regrowth QTL were associated with rhizome and tiller QTL, suggesting all three may be needed for perenniality in Sorghum. Two of the rhizome QTL closely correspond to Rhz2 and Rhz3 found in rice, as mentioned previously (Hu et al. 2003).

Two overwintering QTL have been identified and mapped in an F4 heterogeneous family population from S. bicolor/S. propinquum; one of these QTL overlaps with one of the rhizome QTL just described (Washburn et al. 2013). All of the plants that successfully overwintered had rhizomes; however, only 33% of the overwintering

variation could be explained by the presence of rhizomes. The authors suggested this may be due to either (1) rhizomes not being as important to the overwintering phenotype as previously thought, or (2) current rhizome phenotyping methods are not capable of fully describing rhizome traits. Both Paterson et al. (1995) and Washburn et al. (2013) measured rhizome traits using above-ground indicators; for example, rhizome number was estimated by counting the number of above-ground shoots produced from rhizomes.

This method does not detect rhizomes that did not produce a shoot and does not measure rhizome depth.

Arabidopsis

A single gene, PERPETUAL FLOWERING 1 (PEP1) is the primary regulator of perennial flowering in Arabis alpina, a close perennial relative of Arabidopsis thaliana.

PEP1 is an ortholog of the A. thaliana gene FLOWERING LOCUS C (FLC); it maintains polycarpy and initiates flowering after vernalization (Wang et al. 2009). However, vernalization does not seem to be required for perenniality in A. alpina since five independent mutations in PEP1 have been found in five different accessions, all of which do not require vernalization to flower (Albani et al. 2012). Interestingly, most of the

PEP1 alleles have a tandem arrangement of a full-length and a partial copy of PEP1, which produce two differentially expressed full-length transcripts. Albani et al. (2012) speculate the complex transcriptional pattern of the locus may allow it to participate in more regulatory pathways, which gives rise to the complex phenotype of perenniality.

Present study

In this study, our objectives were (1) to create a linkage map for the F2 population produced after crossing maize B73 and Z. diploperennis, (2) to identify and map QTL responsible for the perennial phenotype, and (3) to identify single nucleotide polymorphism (SNP) markers associated with perennial traits, including overwintering, observed in F2 plants. We used genotyping-by-sequencing (GBS) technology to generate

SNP markers for both parents and a subset of F2 plants to create a linkage map and then used QTL mapping to identify significant relationships between perennial traits and particular SNPs.

CHAPTER 2

MATERIALS AND METHODS

Plant materials

Zea diploperennis (PI 462368; obtained from the North Central Regional Plant

Introduction Station, USDA-ARS in Ames, Iowa) was used as the paternal parent and crossed with maize B73. Eight F1 plants were grown and sib crossed via open pollination.

On May 8, 2014, we planted 800 F2 kernels, of which 482 survived to the end of the phenotyping period. We carried out phenotypic work on all 482 plants, and did genotypic work on a subset of 93 plants in addition to the parents. We also planted eight Z. diploperennis (also PI 462368) from seed in the field to record phenotypic characterizations that would be representative of the Z. diploperennis parent in its first growing season. However, all eight plants were shaded out by neighboring F2 plants 4-6 weeks after planting. They did not show meaningful above-ground biomass until after cutting in September 2014. Two ramets of the Z. diploperennis parental genotype that were transplanted as fully mature plants in an adjacent field the previous year were used for phenotypic characterization as well. Unfortunately, information on Z. diploperennis trait development during the first year of growth could not be provided by these ramets, since they were in their second growing season. B73 plants were also planted in several places in the field to note the flowering time. B73 does not form tillers, produces 14-16 rows of kernels, and dies rapidly after seed set.

Field design

Kernels were planted in one of three blocks in a field (Figure 1) in Athens,

Georgia. Each block contained Z. diploperennis plants at the northwestern corner—two plants in the western block, four in the middle block, two in the eastern block—with F2 plants planted twelve inches apart in the remaining space. Plants were allowed to open pollinate over the course of the summer.

N 45.8 m

26.7 m

Figure 1. Field design for experiment. Kernels were planted outside in a field behind the UGA greenhouses in Athens, Georgia. The entire field is shown in light green. Plants were only grown in areas marked in dark green.

Soil analysis

Six soil samples were taken in January 2015. Samples were taken in evenly spaced intervals, with three samples taken in the northern half of the field and three samples taken in the southern half; sampling sites were not restricted to planting sites

(dark green area in Figure 1). All samples were taken six inches deep in the soil and allowed to dry overnight before being sent to the Soil, Plant, and Water Lab, which is a

part of the Georgia Cooperative Extension and the Agricultural Experiment Station. A routine soil test was requested, which tested for soil pH, extractable phosphorous, potassium, calcium, magnesium, manganese, and zinc. The results were considered to be acceptable for growing maize in northeastern Georgia (Table 1), according to the Georgia

Cooperative Extension (Kissel & Sonon 2008).

Table 1. Means and standard deviations (s.d.) for phosphorous (P), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), manganese (Mn), and pH levels for six soil samples from field. Soil samples processed by the Soil, Plant, and Water Laboratory of the UGA Ag & Environmental Services Labs (http://aesl.uga.edu/).

P K Ca Mg Zn Mn lbs/acre lbs/acre lbs/acre lbs/acre lbs/acre lbs/acre pH Mean 35 256 2,313 275 7 31 6.1 (s.d.) (22.6) (71.5) (615.8) (75.6) (4.1) (12.8) (0.2)

Phenotypic analysis

Seven traits related to the difference in perennial versus annual habit between Z. diploperennis and maize B73 were analyzed in the parents and 482 F2 plants. We recorded the time tassels, pollen, and silks were visible on plants. This information was recorded weekly throughout the summer until the end of August 2014. All plants were cut back with a machete to about eighteen inches in height over the course of September

2014, after which the total number of tillers was recorded for each plant. A tiller was defined as a lateral branch that connected to the plant via a below-ground node (de Leon

& Coors 2002). Two weeks after cutting plants back, we recorded any signs of regrowth in the form of new, above-ground, green tissue. Four to eight weeks after cutting plants

back, we recorded any signs of plants “staying green,” meaning any above-ground tissue was still green. Stay-green tissues were typically stalks and growth spots at nodes.

Overwintering was scored in the spring of 2015.

Genotypic analysis

Genomic DNA was extracted from leaf tissue of 93 F2 plants as well as from the leaf tissue of the maize B73 parent and from the root tissue of the Z. diploperennis (PI

462368) parent using a modified CTAB protocol (modified from a CIMMYT Applied

Molecular Genetics Laboratory Protocol and based on Saghai-Maroof et al. 1984). All tissues were freeze dried before extraction, except for the maize B73 leaf tissue. All leftover freeze dried samples have been stored for future use. Only a subset of the 482 F2 plants was genotyped due to available funds.

Two phenotypic groups were represented in the 93 F2 plants analyzed (Table 2).

The first was comprised of 44 plants that had completely flowered (produced tassels, pollen, and silks) by the end of August and showed regrowth or stay-green traits or both after cutting. This group is referred to as the “perennial hopefuls” and abbreviated PH.

The second group is comprised of 49 plants that had completely flowered and showed no regrowth or stay-green traits after cutting. These plants also had 3-4 tillers per plant, which was the average number of tillers found in the PH group. This second group of plants is referred to as the “crispy tillers” and abbreviated CT.

Table 2. Labels and phenotypic groups of plants genotyped-by-sequence.

Plant Label Phenotypic Group Maize B73 Parent Zea diploperennis (PI 462368) Parent

CC14-222 Perennial Hopeful CC14-223 Perennial Hopeful CC14-224 Perennial Hopeful CC14-235 Crispy Tillers CC14-246 Crispy Tillers CC14-251 Perennial Hopeful CC14-252 Perennial Hopeful CC14-264 Crispy Tillers CC14-266 Perennial Hopeful CC14-280 Crispy Tillers CC14-284 Crispy Tillers CC14-287 Crispy Tillers CC14-297 Crispy Tillers CC14-299 Crispy Tillers CC14-302 Perennial Hopeful CC14-311 Crispy Tillers CC14-319 Perennial Hopeful CC14-321 Perennial Hopeful CC14-322 Crispy Tillers CC14-326 Crispy Tillers CC14-328 Perennial Hopeful CC14-335 Crispy Tillers CC14-336 Perennial Hopeful CC14-348 Crispy Tillers CC14-355 Crispy Tillers CC14-356 Perennial Hopeful CC14-357 Perennial Hopeful CC14-359 Perennial Hopeful CC14-366 Crispy Tillers CC14-367 Crispy Tillers CC14-381 Crispy Tillers CC14-382 Crispy Tillers

CC14-388 Crispy Tillers CC14-390 Crispy Tillers CC14-394 Crispy Tillers CC14-397 Perennial Hopeful CC14-399 Crispy Tillers CC14-400 Crispy Tillers CC14-404 Crispy Tillers CC14-407 Perennial Hopeful CC14-412 Crispy Tillers CC14-413 Crispy Tillers CC14-418 Perennial Hopeful CC14-419 Perennial Hopeful CC14-420 Perennial Hopeful CC14-427 Perennial Hopeful CC14-432 Perennial Hopeful CC14-439 Crispy Tillers CC14-441 Perennial Hopeful CC14-445 Crispy Tillers CC14-446 Crispy Tillers CC14-450 Perennial Hopeful CC14-458 Perennial Hopeful CC14-475 Perennial Hopeful CC14-477 Crispy Tillers CC14-481 Crispy Tillers CC14-483 Crispy Tillers CC14-485 Perennial Hopeful CC14-488 Crispy Tillers CC14-491 Crispy Tillers CC14-492 Crispy Tillers CC14-494 Perennial Hopeful CC14-496 Crispy Tillers CC14-497 Perennial Hopeful

CC14-505 Perennial Hopeful CC14-506 Crispy Tillers CC14-509 Crispy Tillers CC14-513 Crispy Tillers CC14-521 Perennial Hopeful CC14-526 Crispy Tillers CC14-528 Crispy Tillers CC14-537 Perennial Hopeful CC14-539 Crispy Tillers CC14-540 Perennial Hopeful CC14-541 Perennial Hopeful CC14-544 Perennial Hopeful CC14-545 Perennial Hopeful CC14-549 Crispy Tillers CC14-567 Perennial Hopeful CC14-578 Crispy Tillers CC14-580 Crispy Tillers CC14-583 Perennial Hopeful CC14-589 Crispy Tillers CC14-593 Perennial Hopeful CC14-600 Crispy Tillers CC14-607 Perennial Hopeful CC14-626 Crispy Tillers CC14-645 Perennial Hopeful CC14-653 Perennial Hopeful CC14-654 Perennial Hopeful CC14-655 Perennial Hopeful CC14-656 Perennial Hopeful CC14-658 Crispy Tillers

All DNA samples were sent to the Institute for Genomic Diversity (Cornell

University, Ithaca, NY, USA) for 96-plex genotyping-by-sequencing (GBS) analysis using the ApeKI restriction enzyme and following the GBS protocol (Elshire et al. 2011).

Sequencing data analysis and SNP calling

Single nucleotide polymorphism (SNP) calls were made using the raw Illumina sequencing from the Institute for Genomic Diversity and custom scripts from the Devos

Lab at the University of Georgia. Reads were aligned to the maize B73 reference genome

(AGP version 3.25) before filters were used to discard SNPs that were not found in more than 85% of individuals, segregated distortedly due to technical reasons (deviated significantly from the expected Mendelian segregation ration of 1:2:1; determined using

Chi-squared test, p > 0.05), had less than four reads per SNP per individual, or were not homozygous for one parent or the other. Originally, we had wanted eight reads per SNP per individual to be confident in heterozygous calls, but this stringency left too few SNPs to map with.

Genetic map construction

After filtering, the remaining SNPs were mapped using MapMaker 3 (Lander et al. 1987; Paterson et al. 1988; Lincoln et al. 1993; Lincoln 1992).The order of SNPs was based on their position in the B73 reference genome and recombination frequencies were determined using MapMaker 3. MapChart 2.2 (Voorrips 2002) was used to draw the genetic map. Distances between SNPs were determined in centimorgans (cM) using the

Kosambi function. All markers were named with the prefix “M” (for “marker”).

Quantitative Trait Locus/Loci (QTL) mapping

Composite interval mapping (CIM) was done using the computer program QTL

Cartographer for Windows (version 2.5_011, Wang et al. 2012) to map QTL controlling the perennial phenotype in the F2 population. CIM was run with the default program setting for model 6 (5 background markers and a window size of 10 cM). LOD thresholds were determined from 500 permutations of the data at a walk speed of 2 centimorgans

(cM) with the significance threshold set to 0.05 before mapping.

CHAPTER 3

RESULTS

Phenotypic characterization

Flowering

Tassel emergence, pollen shedding, and silks emergence were monitored starting after F2 plants germinated. Flowering abruptly started nine weeks after planting, which is approximately the time maize B73 begins flowering in Athens, Georgia. F2 plants flowered until the end of August (week 15), which is when flowering traits were no longer recorded because the plants had started to show signs of Southern corn rust blight infection, which was particularly severe in 2014. We calculated a histogram, the mean values with standard deviations, and 90% confidence intervals to describe the distribution and variation in the flowering phenotypes (Figure 2, Table 3). The mean range for flowering for the F2 population was from 10.0 to 11.9 weeks, with missing data not included in statistical analysis. Instances of staggered flowering were observed, meaning a plant produced tassels on one stalk and then produce more a few weeks later on another stalk, if present, for example. This phenotype was not formally recorded though. Non- synchronous flowering between the male (tassels) and female (silks) of the same plant was also observed, though not recorded, for some F2 plants. The maize B73 and Z. diploperennis parents do not have mistimed flowering but instead coordinate tassel emergence, pollen shedding, and silks emergence in a concerted manner. For the 93 F2

plants that were genotyped, the distribution and mean values for tassel emergence, pollen shedding, and silk emergence dates were calculated (Figure 4). Mean values and standard deviations were comparable to those of the whole population.

250 B73 200

150

Number 100

50 Dip

0 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Never Time after Planting (weeks)

Tassel emergence Pollen shedding Silks emergence

Figure 2. Distribution of flowering dates for 482 F2 plants. Tassel emergence, pollen shedding, and silks emergence dates were recorded until the last week of August 2014 (week 15). The typical flowering cycle duration for the maize B73 parent is 10 weeks in Georgia. Z. diploperennis flowers in a day length dependent manner, and in Athens, Georgia it occurs in the second week of October. Parental flowering times (B73, Dip) are indicated with horizontal brackets. Only the first exhibition of these traits was recorded for each F2 plant; no cyclical or multiple flowering events were recorded, although they did occur in some plants. Plants that did not exhibit these traits for the first time before the end of phenotyping were categorized as Never.

Table 3. Means, standard deviations (s.d.), and confidence intervals for flowering and tiller traits for 482 F2 plants along with observed values for the eight Z. diploperennis individuals and maize B73 parent. Missing data was not included in the F2 population statistical analysis.

Observed Values Mean (s.d.) Z. 90% Confidence Phenotype Maize B73 F Population diploperennis† 2 Interval Tassel emergence N/A 10 10.0 (1.2) ±0.1 Pollen shedding N/A 10 11.2 (1.2) ±0.1 Silks emergence N/A 10 11.9 (1.3) ±0.1 Tiller number 3-15 0 2.8 (2.1) ±0.2 †Flowering was only observed on the second-year Z. diploperennis ramets and the exact dates were not recorded.

Tiller number

The number of tillers was recorded immediately after plants were cut back in

September 2015. Tillers were defined as lateral branches that connected to the plant at below-ground nodes (de Leon & Coors 2002). To describe the distribution and variation of the tiller number phenotype, we calculated a histogram, mean values with standard deviations, and 90% confidence intervals (Figure 3, Table 3). The mean number of tillers for the F2 population was 2.8, which is between the parental values but skewed towards the maize B73 value of zero. Our F2 mean is similar to those reported by Srinivasan and

Brewbaker (1999). However, there was a significant difference in tiller number between plants in the eastern most planting group and plants in the western most planting group in the field (p < 0.05), with more tillers per plant observed in the eastern group. One reason for this effect may be that the tiller phenotype is environmentally influenced in grass species (Whipple et al. 2011). The two Z. diploperennis parental genotype ramets had an observed 16 to 18 tillers, but they were not in their first year of growth. The eight Z.

diploperennis individuals planted as seed in the spring of 2015 had a mean tiller number of 6.4 (s.d. ±3.8). While these plants are also a weak depiction of the Z. diploperennis first-year phenotype since all eight plants were shaded out until cutback, they are most likely a truer representation than the Z. diploperennis ramets. For the 93 F2 plants that were genotyped, the distribution and mean values for tiller number were calculated

(Figure 4). Mean values and standard deviations were comparable to those of the whole population.

120 DipI

100 B73

60 Number 40

20 DipR

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 No data Tillers

Figure 3. Distribution of tiller values for 482 F2 plants. The values represent the number of tillers in addition to the main stalk of the plant. The typical values for the maize B73 parent (B73), Z. diploperennis individuals (DipI), and Z. diploperennis ramets (DipR) are indicated with horizontal brackets. Tillers were defined as lateral branches that connected to the plant at below-ground nodes (de Leon & Coors 2002).

50 40

A 40 B

30 30 20

Number Number 10 10 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time of Tassel Emergence after Time of Pollen Shedding after Planting (weeks) Planting (weeks)

40 40

C 35

30 D 30 25 20 20

15 Number 10 Number 10 5 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time of Silk Emergence after Planting Tillers (weeks)

Figure 4. Distribution of tassel emergence, pollen shedding, silk emergence, and tiller number values for 93 F2 plants that were genotyped (parents excluded). (A) The mean tassel emergence date among the genotyped F2 plants was 9.8 weeks (s.d. ±0.9). (B) The mean pollen shedding date was 11.1 weeks (s.d. ±1.1). (C) The mean silk emergence date was 12.3 weeks (s.d. ±1.3). All three flowering traits were more representative of the maize B73 parent within the genotyped sample population. (D) The mean tiller number was 3.5 tillers (s.d. ±1.5), which is approximately equidistant between the maize B73 value (0 tillers) and the Z. diploperennis first-year growth mean value (individuals planted from seed; 6.4 tillers).

Regrowth, stay-green, and overwintering

Six F2 plants, all eight Z. diploperennis individuals, and both Z. diploperennis ramets showed regrowth two weeks after being cut back in September 2014 (Table 4).

Forty-three F2 plants showed signs of stay-green tissue—typically stalks and above- ground nodes—four to eight weeks after cutback (Figure 5). One plant, CC14-223, exhibited both regrowth and stay-green traits. Regrowth and stay-green traits were only

considered legitimate if plants completed flowering and set seed before cutback. Annual species usually die shortly after setting seed; recording regrowth and stay-green traits in completely flowered and seeded plants helped ensure non-annual traits were studied.

None of the 482 F2 plants showed regrowth in the spring of 2015 (Table 4). One of the eight Z. diploperennis individuals planted from seed (Dip #2) showed regrowth beginning in mid-March. Both Z. diploperennis ramets of the parental genotype showed regrowth at the same time. From October 2014 to mid-April 2015, there were 61 days with minimum air temperatures at or below freezing, the lowest of which was -12.1 °C on

January 8, 2015. There was only one day the maximum temperature did not get above freezing (Figure 6). No soil temperature data was recorded.

A B

Figure 5. Regrowth and stay-green phenotypes in F2 plants after cutback. (A) An F2 plant shows instances of regrowth (arrowheads) two weeks after cutback. (B) The F2 plant on the left shows no signs of the stay-green trait, whereas the F2 plant on the right has green stalks one month after cutting and is an example of the stay-green phenotype. There were two rounds of cutting and both occurred in September 2014.

Table 4. Fall regrowth and overwintering scores for parents, 482 F2 plants, and Z. diploperennis controls.

Plant Fall Regrowth Spring Regrowth (Overwintering) Maize B73 None None Z. diploperennis ramets Yes Yes

F2 plants Yes (7 of 482) None Z. diploperennis individuals Yes Yes (1 of 8)

C) ° 15

5 Air Temperature ( Temperature Air

2014 2014 2014

2014 2014

2015 2015

2014 2014

2015 2015

2015 2015 2015

- - -

- -

-5 -

Jan Jan

Oct Oct Oct

Apr

Feb Feb

Dec Dec

- -

Nov Nov

Mar Mar

- - -

- -

14 29

16 31

13 28

15 30

15 30 30 -10 15

-15 Date

Maximum Minimum

Figure 6. Maximum and minimum air temperatures from October 2014 to mid-April 2015. Weather data is for the location of Watkinsville, Georgia and is recorded by the Georgia Automated Environmental Monitoring Network (weather.uga.edu).

Genotypic characterization

Genotyping-by-sequencing (GBS) of the 93 F2 plants and the two parents produced a total of 1.65x108 100-nt reads that passed all quality controls, with an average of 1,738,079 reads per barcode. One sample, CC14-246 of the Crispy Tillers (CT) phenotypic group, failed to generate reads. For the Z. diploperennis parent, a total of

3,345,166 100-nt reads were generated, which is approximately twice as many reads as any other sample. Since the Z. diploperennis parent is from a wild accession and therefore most likely heterozygous throughout much of the genome, we asked for extra read coverage to help ensure heterozygous SNPs could be called confidently.

A total of 7,668 SNPs were originally found after aligning sequence reads to the maize B73 genome and removing SNPs that either had fewer than four reads per SNP per individual or were not homozygous in one of the two parents. After further filtering to remove SNPs that were either missing in more than 15% of individuals or exhibiting high, non-biological segregation distortion, a total of 858 SNPs that were homozygous for contrasting alleles in either parent remained. These 858 SNPs were used to generate a genetic map (Figure 7, Figure 8).

The limited size of the genotyped samples (n = 93) and the occurrence of only two meioses (male and female) before production of the F2 mapping population meant a restricted number of recombination events was able to be observed. The 858 SNPs were distributed across all ten chromosomes, ranging from 49 (chromosome 7) to 120 SNPs

(chromosome 1) with an average of 85.8 SNPs per chromosome (Table 5). The mean distance between SNPs for each chromosome ranged from 1.1 to 2.6 centimorgans (cM) with an average of 1.7 cM for all chromosomes, resulting in a mean density of 0.6 SNPs

per cM for the genome. Chromosome 9 had the smallest distance between markers (1.1 cM) and the highest marker density (0.9 SNPs/cM), while chromosome 6 had the largest mean distance between markers (2.6 cM) and the lowest marker density (0.4 SNPs/cM).

Segregation distortion due to biological factors was found, with 17.4% of SNPs (149 out of 858 SNPs) showing significant distortion (p < 0.01). A particularly large distortion region with all loci distorted towards the maize B73 alleles was found on chromosome 5 from position 10,224,187 to 216,043,427 bp (from marker M391 to M477), relative to the maize B73 reference genome (Table 6). Of the 100 SNP markers on chromosome 5, 76% showed very significant distortion (p < 0.001). The total length of the genetic map was

1,437.5 cM, which is 16.8% shorter than the genetic map for an F2 mapping population with a similar marker density from two maize parents generated by Davis et al. (1999)

(Table 5). The ten chromosomes had fairly good coverage and uniformity of marker distribution, with the largest distance between markers being 20.9 cM. The resulting map had no interval greater than 21 cM in length, and only 18 intervals were larger than 10 cM (Figure 7, Figure 8).

Table 5. Number of SNPs, map length, mean distance between SNPs, and density of SNPs per chromosome for genetic map of F2 hybrids between maize B73 and Z. diploperennis. For map length comparison, genetic map length data from a maize x maize F2 population is shown (Davis et al. 1999).

Mean distance Length Maize- Number of Density Length B73-Dip Chrom. between SNPs Maize Davis et al. SNPs (SNP/cM) Coatney (cM) (cM) (cM) 1 120 1.6 0.6 192.7 245.2 2 102 1.5 0.7 155.6 200.2 3 85 2.0 0.5 166.6 164.8 4 69 2.0 0.5 138.8 169.7 5 100 1.5 0.7 150.3 174.8 6 49 2.6 0.4 126.0 168.6 7 90 1.7 0.6 152.9 147.5 8 74 1.7 0.6 123.6 167.6 9 100 1.1 0.9 111.4 150.4 10 69 1.7 0.6 119.6 138.6 Total 858 1.7 0.6 1,437.5 1,727.4

Table 6. Chi-squared significance values for segregation patterns of SNPs across all chromosomes. SNPs with no significance segregated in a Mendelian manner, whereas SNPs with greater statistical significance segregated in a distorted manner since they deviated from expected Mendelian segregation ratios (1:2:1). The observed segregation distortion was most likely due to biological factors (SNPs distorted due to technical factors were removed).

# SNPs with # SNPs with # SNPs with # SNPs with no significance significance significance Chrom. Total significance of of of p < 0.05 p < 0.01 p < 0.001 1 118 2 0 0 120 2 80 7 8 7 102 3 76 8 1 0 85 4 40 22 6 1 69 5 14 4 6 76 100 6 48 1 0 0 49 7 48 29 12 1 90 8 36 24 11 3 74 9 58 27 10 5 100 10 50 17 2 0 69 Total 568 141 56 93 858 Percentage 66.3% 16.4% 6.5% 10.8% 100.0%

Chrom1 Chrom2 Chrom3 Chrom4 Chrom5 Chrom1 Chrom2 Chrom3 Chrom4 Chrom5 0.0 M1 0.0 M377 1.4 M2 0.0 M121 M378 3.4 M3 M122 M123 2.6 M379 M380 7.3 M4 5.5 M124 4.5 M381 M382 12.3 M5 M125 M126 8.8 M383 15.3 M6 M7 11.4 M127 9.7 M384 M385 M8 M9 13.9 M128 20.5 17.2 M386 M10 M11 M129 M130 0.0 M223 M387 26.6 M12 M13 20.6 M131 7.7 M224 19.4 21.6 M388 27.8 M14 22.2 M132 15.5 M225 25.0 M389 36.2 M15 M16 28.2 M133 M226 M227 26.8 41.3 M390 37.9 M17 31.7 M134 M228 0.0 M308 M309 44.2 M391 42.7 M18 32.6 M135 34.1 M229 12.2 M310 M311 46.4 M392 M19 M20 35.6 M136 M137 40.6 M230 45.8 15.8 M312 49.1 M393 M21 37.5 M138 M139 44.1 M231 17.0 M313 49.8 M394 47.0 M22 39.7 M140 M141 45.4 M232 18.4 M314 54.8 M395 51.4 M23 42.4 M142 M143 45.7 M233 19.5 M315 57.2 M396 M397 54.6 M24 M25 44.9 M144 49.4 M234 M235 27.8 M316 62.1 M398 65.3 M26 M27 45.5 M145 M146 50.1 M236 37.5 M317 M318 M399 67.4 M28 M147 M148 50.6 M237 62.9 47.8 M319 M320 M400 M29 M30 46.0 M149 52.9 M238 M239 63.7 71.5 54.2 M321 M322 64.9 M401 M31 47.1 M150 54.3 M240 59.1 M323 M324 68.4 M402 77.0 M32 M33 48.1 M151 M241 M242 60.1 M325 M326 M403 M404 77.8 M34 M35 M152 M153 M243 M244 69.0 61.2 M327 M405 M406 78.5 M36 M37 51.4 M154 57.7 M245 M246 70.1 62.1 M328 M407 M408 79.2 M38 M39 54.3 M155 M156 M247 M248 M329 M330 71.1 M409 M410 82.1 M40 55.0 M157 M249 M250 65.8 M331 M332 M411 M412 85.2 M41 M42 56.6 M158 58.4 M251 M333 72.2 M413 M414 M43 M44 58.8 M159 M160 60.3 M252 86.6 67.4 M334 M335 M415 M45 M161 M162 M253 M254 M336 M337 M416 M417 88.9 M46 62.3 M163 M164 M255 M256 70.1 61.7 M338 72.7 M418 90.4 M47 65.5 M165 M166 M257 M258 74.9 M339 74.6 M419 M420 91.5 M48 M49 67.3 M167 M168 M259 M260 80.5 M340 M341 75.1 M421 M422 94.1 M50 M51 67.5 M169 64.4 M261 M262 81.8 M342 M423 M424 95.0 M52 M170 M171 65.3 M263 M264 85.9 M343 76.7 M425 96.5 M53 M54 67.8 M172 M173 70.0 M265 90.8 M344 M426 M427 99.0 M55 M56 M174 76.4 M266 78.9 97.4 M345 79.5 M428 101.6 M57 69.4 M175 M176 77.5 M267 101.9 M346 M347 81.5 M429 M430 104.7 M58 M59 71.6 M177 78.1 M268 102.7 M348 M431 M432 106.2 M60 75.6 M178 M179 M269 M270 78.9 104.2 M349 M350 83.5 M433 M434 107.6 M61 76.4 M180 M181 M271 106.3 M351 M352 M435 110.6 M62 M63 78.3 M182 M183 79.5 M272 107.3 M353 M354 87.0 M436 112.4 M64 79.7 M184 M185 89.2 M273 109.1 M355 88.2 M437 113.8 M65 M66 82.7 M186 105.9 M274 113.4 M356 89.3 M438 116.5 M67 83.6 M187 M188 106.8 M275 116.0 M357 M439 M440 126.4 M68 90.0 M189 107.4 M276 M358 M359 90.0 M441 M442 127.6 M69 92.9 M190 111.3 M277 M278 118.7 M360 90.6 M443 129.4 M70 95.0 M191 M192 111.8 M279 M361 M362 92.2 M444 129.9 M71 M72 96.1 M193 117.7 M280 M281 121.3 M363 M445 M73 M74 M194 M195 118.8 M282 M283 93.3 122.6 M364 M365 M446 M75 M76 96.7 M196 122.8 M284 95.3 123.1 M366 M367 M447 M448 M77 M78 97.9 M197 123.7 M285 130.9 124.7 M368 M369 99.3 M449 M79 M80 98.5 M198 124.5 M286 124.8 M370 100.9 M450 M451 M81 M82 99.1 M199 125.9 M287 125.3 M371 M452 M453 M83 101.2 M200 M288 M289 129.3 126.5 M372 M373 104.2 M454 131.9 M84 M85 103.6 M201 M202 M290 131.4 M374 106.7 M455 M456 133.8 M86 M87 107.0 M203 M204 132.9 M291 135.3 M375 108.5 M457 M458 M88 M89 114.8 M205 135.1 M292 M293 135.0 137.4 M376 109.9 M459 M460 M90 115.8 M206 150.9 M294 M461 M462 M91 M92 117.2 M207 M295 M296 135.5 151.6 112.8 M463 M93 M94 118.9 M208 M297 113.8 M464 M465 M95 M96 123.1 M209 156.3 M298 136.0 114.8 M466 M97 M98 127.8 M210 156.8 M299 M300 114.9 M467 136.6 M99 M100 134.8 M211 158.8 M301 M302 117.7 M468 M101 M102 137.1 M212 161.9 M303 M304 137.7 126.7 M469 M470 M103 141.8 M213 163.0 M305 M306 M471 M472 141.3 M104 147.7 M214 165.0 M307 131.3 M473 146.2 M105 148.9 M215 M216 134.4 M474 149.0 M106 M107 150.0 M217 M475 157.9 M108 M218 M219 137.8 148.2 M477 158.8 M109 154.0 M220 M221 159.6 M110 M222 160.7 M111 M112 161.9 M113 M114 165.5 M115 M116 180.5 M117 M118 M119 190.0 M120

Figure 7. Genetic map for chromosomes 1-5 for F2 mapping population. Numbers to the left of each chromosome are cumulative distances for each SNP marker (cM). Marker M389, which was associated with a stay-green QTL, on chromosome 5 is indicated in bold green text. Each marker is labeled MXXX on the right of each chromosome.

Chrom6 Chrom7 Chrom8 Chrom9 Chrom10 Chrom6 Chrom7 Chrom8 Chrom9 Chrom10 0.0 M691 M692 M693 M694 M695 M696 M697 M698 1.3 M699 M700 0.0 M527 M701 M702 8.6 M528 M529 M703 13.7 M530 M531 2.2 M704 M705 16.0 M532 0.0 M617 M706 M707 0.0 M791 M533 M534 M618 M619 15.4 3.1 M708 M709 18.3 M792 21.5 M535 M620 M710 M711 22.0 M793 27.6 M536 M537 19.9 M621 3.8 M712 M713 M794 M795 35.5 M538 21.1 M622 M623 23.3 M714 M715 M796 43.3 M539 M540 24.1 M624 6.2 M716 25.2 M797 46.6 M541 25.0 M625 M798 M799 7.8 M717 M718 28.6 47.4 M542 26.6 M626 M800 9.8 M719 M720 34.2 M478 48.7 M543 M544 28.0 M627 M801 M802 0.0 11.1 M721 35.3 M479 M480 49.4 M545 M628 M629 M803 M804 29.4 12.3 M722 15.1 51.6 M546 M547 M630 36.3 M481 M482 M723 M724 M805 M806 M548 M549 35.5 M631 M632 15.5 M483 M484 14.1 M725 M726 38.2 M807 M808 M485 52.2 M550 M551 36.8 M633 M809 M810 16.5 15.4 M727 M728 39.5 M486 M487 M552 37.3 M634 M635 M811 16.7 M729 M730 40.5 17.8 52.7 M553 M554 M636 M637 M488 M489 38.8 M731 M732 M812 M813 55.4 M555 M556 M638 42.4 27.0 M490 M491 18.6 M733 M814 M492 M557 M558 39.4 M639 M640 M815 29.1 57.6 21.0 M734 M735 44.6 M493 M494 M559 41.6 M641 M642 M816 M817 31.0 21.7 M736 46.7 M495 M496 58.2 M560 M643 M644 M818 34.2 45.6 23.1 M737 M738 50.6 M497 58.8 M561 M562 M645 M819 36.0 25.2 M739 M740 51.4 64.6 M563 46.1 M646 M647 38.3 M498 M741 M742 53.1 M820 M564 M565 48.5 M648 M649 28.1 43.1 M499 M500 M743 54.2 M821 M822 67.2 M566 51.0 M650 M651 28.7 48.3 M501 M744 M745 M823 M824 70.9 M567 M568 M652 M653 52.3 M502 52.1 29.8 M746 57.3 M825 M826 M503 77.9 M569 M570 M654 M655 M827 53.2 30.2 M747 M748 81.7 M571 M572 55.1 M656 55.9 M504 M505 M749 M750 59.7 M828 M829 83.0 M573 56.4 M657 60.2 M506 31.1 M751 M830 M831 M507 M508 85.5 M574 M575 58.2 M658 M659 61.3 M832 61.8 34.0 M752 M509 M576 M577 58.7 M660 M833 62.6 88.7 36.0 M753 M754 61.9 M510 M511 M578 59.3 M661 M662 M834 M835 67.4 38.8 M755 M756 M512 90.6 M579 62.6 M663 M664 M836 M837 73.6 41.5 M757 62.5 M513 101.5 M580 M581 66.7 M665 M838 83.8 42.7 M758 M759 M514 M582 M583 68.1 M666 M667 M839 88.4 45.0 M760 M761 63.2 M515 M516 M584 M585 69.5 M668 65.4 M840 M841 89.0 102.7 47.2 M762 M763 M517 M518 M586 M587 70.9 M669 M670 M842 48.1 M764 66.3 M519 M588 71.9 M671 M672 M843 89.6 51.5 M765 M766 73.3 M520 105.9 M589 72.3 M673 M844 M845 91.3 55.9 M767 M521 M522 106.8 M590 M591 73.1 M674 79.7 M846 94.8 56.7 M768 109.0 M592 79.1 M675 115.7 M523 M769 M770 M847 M848 110.6 M593 M594 83.3 M676 82.8 119.0 M524 60.7 M771 M849 M525 112.5 M595 M596 83.9 M677 M678 M850 120.9 63.0 M772 87.3 M526 112.8 M597 90.4 M679 M851 M852 125.2 64.0 M773 M774 89.6 112.9 M598 94.8 M680 M853 65.2 M775 90.3 113.5 M599 M600 97.0 M681 M682 M854 68.0 M776 M777 92.7 114.8 M601 99.4 M683 M855 70.1 M778 111.7 119.3 M602 109.5 M684 M856 71.0 M779 112.3 120.8 M603 M685 M686 M857 114.0 73.9 M780 113.1 122.4 M604 M687 M858 77.8 M781 115.0 123.8 M605 117.5 M688 M782 M783 118.8 M859 126.2 M606 122.5 M689 M690 83.6 M784 136.3 M607 85.9 M785 142.3 M608 86.7 M786 M787 143.0 M609 104.7 M788 145.2 M610 M611 108.2 M789 147.4 M612 M613 109.8 M790 150.2 M614 M615 151.5 M616

Figure 8. Genetic map for chromosomes 6-10 for F2 mapping population. Numbers to the left of each chromosome are cumulative distances for each SNP marker (cM). Each marker is labeled MXXX on the right of each chromosome.

Quantitative Trait Locus/Loci (QTL) mapping

Composite interval mapping (CIM) found a single QTL for one of the seven perennial traits scored. With a LOD threshold of 4.1, a single QTL for the stay-green

phenotype was found on chromosome 5, which was associated with marker M389

(position 5:6364831on maize B73 reference genome) (Figure 9). M389 is located near the end of the short arm of chromosome 5 in maize B73 and in this particular F2 mapping population was close to a recombination break point. While M389 was closely associated with the stay-green QTL peak, neighboring markers M388 and M390 also supported the peak with high, but not significant, LOD scores and were seen as shoulder peaks on the

LOD graph (Figure 9). The physical distance between neighboring markers M388 and

M390 on the reference genome is 5.29 Mbp, which covers approximately 443 protein coding genes (www.maizegdb.org). Interestingly, when only considering marker M389, the stay-green plants showed expected segregation ratios at this locus while the non-stay- green plants had distorted segregation towards the maize B73 allele (p < 0.001). The stay- green QTL explained 27.0% (r2) of the total phenotypic variation. The additive and dominant effect values were -0.34 and 0.12, respectively, which suggested Z. diploperennis alleles contributed more positively to the effect than the corresponding maize B73 alleles (Table 7). No other significant QTLs were found for any of the other recorded traits.

32 Chrom5

0.0 M377 2.6 M378 4.5 M379 M380 8.8 M381 M382 9.7 M383 M384 M385 17.2 M386 19.4 M387 21.6 M388 25.0 M389 41.3 M390 44.2 M391 46.4 M392 49.1 M393 49.8 M394 54.8 M395 57.2 M396 M397 62.1 M398 62.9 M399 63.7 M400 64.9 M401 0 2 4 6 8 68.4 M402 69.0 M403 M404 70.1 M405 M406 M407 M408 71.1 M409 M410 stay-green M411 M412 72.2 M413 M414 M415 M416 M417 72.7 M418 74.6 M419 M420 75.1 M421 M422 M423 M424 76.7 M425 78.9 M426 M427 79.5 M428 81.5 M429 M430 M431 M432 83.5 M433 M434 M435 87.0 M436 88.2 M437 89.3 M438 M439 M440 90.0 M441 M442 90.6 M443 92.2 M444 93.3 M445 95.3 M446 M447 M448 99.3 M449 100.9 M450 M451 M452 M453 104.2 M454 106.7 M455 M456 108.5 M457 M458 109.9 M459 M460 M461 M462 112.8 M463 113.8 M464 M465 114.8 M466 114.9 M467 117.7 M468 126.7 M469 M470 M471 M472 131.3 M473 134.4 M474 137.8 M475 148.2 M477

Figure 9. Genetic map of chromosome 5 plotted with QTL interval and QTL LOD graph for 93 F2 plants. The QTL interval, represented vertically in green, is shown with 1 and 2 LOD intervals to the right of the map. The QTL LOD graph shows the LOD threshold of 4.1 for the stay-green phenotype. Marker M389, the most significantly associated marker with the QTL peak, is shown in bold green text.

Table 7. Chromosome location and direction of QTL for stay-green phenotype estimated by composite interval mapping (CIM) in 93 genotyped F2 plants. MML nearest molecular marker loci, a additive effect, d dominance effect, Dir direction of the effect (whether the M maize or D Z. diploperennis allele contributed positively to the effect), r2 proportion of phenotypic variation explained by QTL.

Chromosome Position (cM) MML LOD a d Dir r2 5 0.2501-0.4101 M389 6.9 -0.34 0.12 D 0.27

CHAPTER 4

DISCUSSION AND CONCLUSIONS

Failure to overwinter

Zero F2 plants, one Z. diploperennis individual planted from seed, and both Z. diploperennis adult ramets transplanted to the field two years ago overwintered. It is unclear why the overwintering rate for the Z. diploperennis individuals was low while the adult ramets have successfully done so the past two years. It is known that rhizome development distinguishes perennial Zea species from their annual counterparts

(Westerbergh & Doebley 2004). The data reported here raises questions about differences in rhizome development between first-year Z. diploperennis plants and older plants, assuming the shade effect the first-year plants experienced is not to blame. For example, did the shorter growing season in Athens, Georgia (relative to the native environment in central Mexico) stunt rhizome formation and thus hamper perenniality in the first-year plants? The adult plants were transplanted from the greenhouse as adults and did not experience a stunted growing season during the first year. Another factor to consider is if first-year rhizomes have different capabilities than older rhizomes. The potential for nutrient storage capacity may change with age. While differences in storage capacity might not impact Z. diploperennis in its native, tropical environment, these differences might influence the plant at northern latitudes. Rhizome depth has been shown to be important for successful overwintering for Sorghum in cold environments (Warwick et al.

1984; Warwick et al. 1986). Rhizome depth may increase with age, which would explain why the majority of the first-year plants failed to overwinter. These questions could easily be answered with a simple trait characterization study for first-year and adult Z. diploperennis plants in the field or greenhouse. Basic rhizome biology data would greatly improve experimental designs for future work similar to that reported here.

As for why none of the F2 plants overwintered, those that produced rhizomes may have had the same fate as the majority of first-year Z. diploperennis individuals discussed earlier. However, additional factors concerning the influence of parental genomes should be considered. The maize B73 genome has experienced significant selection. There is also significant presence-absence variation between maize inbred lines (Springer et al.

2009), which might be further exaggerated between inbred lines and teosinte species.

Together, the high degree of selection and presence-absence variation may cause maize

B73 to lack key genetic factors or to be unable to contribute to important epistatic interactions to make the perennial phenotype possible in hybrid progeny. Maize B73 may be an unsuitable parent for a mapping population focused on uncovering the genetics of perenniality. Inbred maize lines in general may be unsuitable parents considering

Srinivasan and Brewbaker (1999) used eleven different Hawaiian inbreds as mapping parents to no avail. An approach like the one used by Westerbergh and Doebley (2004), where the annual teosinte species Z. mays ssp. parviglumis was crossed with Z. diploperennis, may prove more fruitful (in their study, Westerbergh and Doebley (2004) did not score for overwintering). Indeed, researchers at South Dakota State University have found preliminary success recovering certain perennial traits in F2 and F3

populations after crossing Z. diploperennis with an annual Northern Flint variety (Zea mays cv. Rhee Flint) (Qiu et al. 2015).

Another possibility for the failure of all F2 individuals to overwinter is the phenotypic threshold for overwintering was not met in any of the plants. Overwintering, while scored phenotypically as a binary trait (yes/no), is most likely a very quantitative trait genotypically and therefore considered a threshold trait. A certain genotypic threshold must be met for the overwintering phenotype to be present. It is possible the threshold was not met for any of the F2 individuals; segregation distortion might have prevented important overwintering loci to be sufficiently assembled. The segregation distortion region on chromosome 5 completely distorted towards maize B73 alleles, for example.

Segregation distortion

Of the 858 SNP markers used to make the genetic map, 17.4% showed significant segregation distortion (p < 0.01). Segregation distortion has been found in a previous genetic map for a BC1 population generated from a cross between maize B73 and Z. diploperennis. Wang et al. (2012) found 144 out of 320 (45%) simple sequence repeats

(SSR) markers segregating distortedly. Specifically on chromosome 5, they found two segregation distortion regions and two gametophytic factors. This phenomenon has been reported before for intraspecies crosses within the Zea genus (Doebley & Stec 1993), as well as in other crop species.

Genetic map

The genetic map reported here has 858 SNPs across ten chromosomes spanning a total of 1,437.5 cM. This is 16.8% shorter than total length reported for a previous map built using an F2 population derived from a cross between two maize inbred lines, Tx303 and CO159, and 1,736 markers of different marker types (Davis et al. 1999). The two maps are reasonably close to each other in length despite one is derived from an interspecies cross and the other from an intraspecies cross. Interspecies crosses typically have lower recombination rates and therefore generate shorter linkage maps.

One limitation to the approach reported here was mapping reads to the maize B73 reference genome. While the reference genome is a powerful resource, important information can be lost during wide hybridization crosses if no other resources are used.

Only reads that matched closely enough with the reference genome were selected for analysis. Therefore, some structural variations in either the Z. diploperennis parent or F2 progeny may have gone undetected based on our approach.

Perenniality genetics

A total of one QTL was detected for traits related to perenniality. None of the flowering traits had any significant QTL peaks, which is consistent with the literature

(Buckler et al. 2009). Tiller number also did not generate any significant QTL peaks, but that was expected since nearly all of the genotyped plants were selected to have 3-4 tillers per plant. Plants that showed regrowth in the fall after cutback did not generate any significant QTL data; no plants successfully overwintered, so there was a dearth of information for this trait as well. However, plants that stayed-green a month after cutback

provided significant QTL data. A peak on chromosome 5 with a peak LOD score of 6.9

(significance threshold of LOD 4.1) was associated with the stay-green phenotype. This peak was linked to SNP marker M389, the thirteenth marker on chromosome 5, and was supported by neighboring markers M388 and M390. The neighboring markers were spaced from M389 by 3.4 cM and 16.3 cM, respectively. Additional markers were found in this region but were excluded from the final analysis due to missing data. Increased read coverage in the future would help resolve the size of this peak.

None of the maize research on perenniality scored for the stay-green trait.

Withered stems, a trait potentially related to the stay-green phenotype, was studied by

Westerbergh and Doebley (2004) in Z. diploperennis and Z. parviglumis F2 progeny four months after planting; however, no phenotyping was done after seed set like in the study presented here. Some research has been done on the hereditary basis of stay-green traits in maize outside the context of perenniality. Fourteen QTL related to the stay-green phenotype were found in F2:3 lines derived from a cross between maize inbred Mo17

(non-stay-green) and maize inbred Q319 (stay-green) using SSR markers and CIM

(Zheng et al. 2009). Stay-green was scored as the percentage of green leaf area present and was measured at different time points after flowering. No QTL mapped to the same area as the stay-green QTL reported here. Before Zheng et al. (2009), Beavis et al. (1994) attempted to map QTL associated with stay-green (among other traits) as a proof-of- concept for using topcrossed and F4 progeny to identify QTL. The way stay-green tissue was measured and scored was not specified. Three QTL were identified using interval mapping, but none were located on chromosome 5 and none were in agreement with those reported by Zheng et al. (2009).

In sorghum, one of maize’s closest relatives, more research has been done to map stay-green QTL. Four stay-green QTL were first identified in 2000 on chromosomes 2, 3, and 5 in an F7 RIL population (Xu et al. 2000). Phenotyping was during post-flowering drought stress with a visual scale of 1 to 5. These QTL were confirmed after more molecular markers were added to the analysis with one QTL, Stg2 (chromosome 3), found to explain the most of the phenotypic variation (Subudhi et al. 2000). Additional stay-green QTL were found by Haussmann et al. (2002) after studying two different recombinant inbred populations and finding only three QTL were consistent between the two different genetic backgrounds over two experimental years. Stay-green tissue was measured in a similar manner to that of Zheng et al. (2009). These QTL were located on chromosomes 1, 7, and 10. None of these QTL studies were studying stay-green phenotypes in a perenniality context. Of the sorghum stay-green QTL mentioned here, only one found by Haussmann et al. (2002) mapped to chromosome 5 in maize. One of the flanking markers for the sorghum QTL was the RFLP marker umc166, which is located on the short arm of maize chromosome 5 and on the top half of sorghum chromosome 1. The stay-green QTL found in the data reported here and the umc166 marker are both located in the same syntenic block between maize and sorghum (Figure

10). The shared homology in this region lends support to the stay-green QTL data found here and invites future investigation.

Figure 10. Synteny between maize B73 chromosome 5 (left) and Sorghum bicolor chromosome 1 (right) at location of stay-green QTL. Red boxes indicate the area between the flanking markers, M388 and M390, of the stay-green QTL on the maize chromosome and the homologous region on the sorghum chromosome; pink boxes highlight syntenic blocks; black and brown lines connect syntenic blocks with the same or reverse, respectively, orientation; yellow stars indicate location of umc166 marker. Image was generated on www.gramene.org with stars added.

There are significant limitations of the QTL analysis reported here. First, no environmental (multiple field locations) or genotypic (clones) replications were used.

These types of replications help control for phenotypic variation that is not specific to genotype. To lend confidence to the data reported here, a follow-up experiment with

added replication—multiple field locations would most likely be easiest to achieve— should be done to see if the same results are produced. Second, the sample size of the experiment limited the findings. In addition to incorporating replication into future experimental designs, the sample size should be significantly increased. While 482 F2 plants were phenotyped, only 93 F2 plants were genotyped. The Beavis effect states that a sample size of 100 will overestimate the phenotypic variance of correctly identified QTL by 10-fold and will only have a 3% chance of detecting small QTL (Xu 2003). However, if 1,000 progeny are analyzed, phenotypic variance estimates are “fairly close” to their true values. Therefore the phenotypic variance reported here for the stay-green QTL is certainly too high. Lastly, the QTL mapping analysis used here is not appropriate for some of the trait data collected. A basic assumption of composite interval mapping

(CIM), as well as for other standard mapping analyses, is that trait data is continuous. The flowering and tiller traits in this study are examples of continuous data. A range, or continuum, of values was possible to describe the phenotypic states. However, the regrowth, stay-green, and overwintering traits were scored as presence/absence traits.

Only two values were used to describe the observed phenotypes and therefore did not generate continuous data. To perform CIM, the presence/absence scores were converted to 1/0 values and classified as continuous data in QTL Cartographer. While there most likely is a significant QTL on chromosome 5 as found with CIM because of its LOD score and presence of supporting neighboring markers, a better estimate of its size and effect could be obtained if a more appropriate analysis was used that took into account the binary nature of the presence/absence traits.

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