IDENTIFICATION AND PERFORMANCE
‘ ‘ or JUGLANS HYBRIDS

Thesis for the Degree of PII. D. ,
‘ MICHIGAN STATE UNIVERSITY
DAVID TRUMAN FUNK
’ 1971 ,

 

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thesis entitled

IDENTIFICATION AND PERFORMANCE OF JUGLANS HYBRIDS

presented by

David Truman Funk

has been accepted towards fulfillment
of the requirements for

_1311_&-__degree in Foregtry

/ Major professor

DaugNovember 11, 1971

 

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l

- ABSTRACT
IDENTIFICATION AND PERFORMANCE OF JUGLANS HYBRIDS
By

David Truman Funk

For many years, the potential timber value of hybrid walnuts has
been acknowledged, but they are hard to produce artificially, rarely
encountered in nature, and difficult to identify with certainty. The
objectives of this study are to relate morphological and chemical char—
acteristics of suspected hybrid walnut seedlings and trees to correspond—
ing characteristics of presumed parent species, and to determine whether
charaCteristics measured in the hybrids are intermediate, exceed
parental values, or perhaps represent unique hybrid traits or combina-
tions of traits.

Twenty—three traits of leaf and branchlet morphology were analyzed
in an attempt to determine those that are most suitable for character—
izing Juglans species and distinguishing hybrids. For several traits in
g, nigra, an analysis of variance indicated a larger component for
leaves within trees than for trees within families or among families.

It appears that it will be necessary to analyze perhaps 3 to 6 leaves
per tree in order to obtain reliable mean values for foliar character-
istics of individual black walnut trees.

The following traits of leaf morphology were selected for distin—
guishing Juglans nigra, g, £2512! and putative hybrids between the two

species: leaflets per leaf, marginal serrations per centimeter,

David Truman Funk

position of the longest leaflet pair on the leaf, leaflet length:width
index, and leaf pubescence. A weighted hybrid index was calculated that
separates Persian from black walnut trees and can be used satisfactorily
to classify most putative hybrids. Variation of hybrid index values
within seedling families of putative hybrid walnut parents is greater
than in pure—species families, an additional confirmation of parental
hybridity.

Other traits such as leaflet length and lopsidedness, rachis
pubescence and branchlet pith color were used to differentiate g,

sieboldiana, g, cinerea, and their hybrids.

 

To supplement the morphological analysis, paper chromatography was
used to analyze Juglans foliage extracts for presence of polyphenols.
Among the several chromatograms prepared, a total of 79 different spots
was distinguished. Some spots tended to be more prevalent in one taxon
than in the others, but the distinction was not usually absolute. In
order to quantify the differences, 'diagnostic values' were developed
for Juglans nigra, g, EEfilé, and their hybrid. The diagnostic values
were computed from a table of decimal fractions indicating the relative
frequency of occurrence of the compounds in all the chromatograms for
each species or the hybrid. The biochemical diagnostic values were well
correlated with morphological hybrid index values; the more expensive
chromatographic techniques will probably be used only when necessary to
resolve doubtful classification.

Measurements of young progeny—test plantations in Michigan and
Illinois suggest that there may be an opportunity to make simultaneous

genetic selection for rapid height growth and improved form in hybrid

David Truman Funk

walnuts. The increased variability in these hybrid populations is
desirable since it is assumed to indicate increased genetic diversity
which in turn allows greater opportunities for selection and tree
improvement. If recombinations yielding improvement in both growth and
form prove to be common among hybrid walnut progeny, an expanded program

of hybrid breeding will be easily justified.

IDENTIFICATION AND PERFORMANCE OF JUGLANS HYBRIDS

by

David Truman Funk

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Forestry

1971

ACKNOWLEDGMENTS

Research described in this thesis was conducted under Cooperative
Agreement Supplement No. 8 to the Master Memorandum of Understanding
of December 6, 1961 between the Forest Service, U.S.D.A. and Michigan
State University.

It is my privilege to acknowledge the countless hours given up by
my wife, Nancy, and daughter, Sara, to data collection, laboratory
work, and neglected family affairs; their patience and good nature
sustained me when I ran short of these virtues.

I appreciate the swift and careful typing of this manuscript by
Martha Dillow, who agreed to 'one more thesis' and kept her promise.

Several tips on chromatographic technique from Professor James
BeMiller of the Department of Chemistry, Southern Illinois University,
and Dr. R. C. Wilkinson of the U.S. Forest service aided me considerably
in completion of the chemical analyses.

The members of my guidance committee, Professor M. Wayne Adams,
Professor August De Hertogh, Professor James W. Hanover, Professor
Stanley K. Ries, and Professor Jonathan W. Wright helped me with
thoughtful advice any time that I had sense enough to ask for it. I
am especially happy to acknowledge the guidance of Professor Wright
who has helped me to appreciate the importance of combining rigor and

practicality in forestry research.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES iv
LIST OF FIGURES vi
CHAPTER
I. HYBRID WALNUTS AND HYBRID ANALYSIS . . . . . . . . . . . l
OBJECTIVES . . . . . . . . . . . . . . . . . . . . 7
II. MORPHOLOGICAL ANALYSES OF WALNUT SEEDLINGS . . . . . . . 8
METHOD 8 O O O O O O O O O O O O O O O O O O O O O O l 0
BLACK WALNUT MORPHOLOGY O O O O O O O O O O O O O O l 3
MORPHOLOGY 0F PERSIAN WALNUT AND PERSIAN X
BLACK WALNUT IIYBRIDS O O O O O O O O O O O O 0 l6
COMPARING SPECIES AND HYBRID POPULATIONS . . . . . l6
Calculating a Hybrid Index
Separation of Hybrids from Parent Species
BUTTERNUT AND JAPANESE WALNUT MORPHOLOGY . . . . . 25
BUTTERNUT X JAPANESE WALNUT HYBRIDS . . . . . . . . 27
BLACK WALNUT HYBRIDS WITH OTHER SPECIES . . . . . . 31
Black Walnut x Butternut Hybrids
Black Walnut x Heartnut Hybrids
III. CHROMATOGRAPHIC ANALYSES OF WALNUT FOLIAGE EXTRACTS . . 36
CHROMATOGRAPHIC TECHNIQUE . . . . . . . . . . . . . 39
PAPER CHROMATOGRAPHY AS AN AID IN IDENTIFYING
PERSIAN X BLACK WALNUT HYBRIDS . . . . . . . . 41
Paired Affinity Index
Diagnostic Values
IV. PLANTATION PERFORMANCE . . . . . . . . . . . . . . . . . 54
V. CONCLUSIONS AND SUGGESTIONS FOR FURTHER RESEARCH . . . . 57
LITERATURE CITED 59
VITA 66
APPENDIX 68

iii

TABLE

1.

2.

lo.

11.

12.

13.

LIST OF TABLES

Hybrid walnuts mentioned in literature . . . . . . . . .
Components of variance for selected traits in Juglans

nigra leaves 0 O O O O O O O O O O I O O O O O I O O O 0

Statistics used in computation of hybrid index for
Juglans regia and J, nigra . . . . . . . . . . . . . .

Range of hybrid index within half-sib walnut families .

Statistics used in computation of hybrid index for
Juglans sieboldiana and J, cinerea . . . . . . . . . . .

Hybrid index comparisons for hybrid walnut seedling
families and assumed parental combinations . . . . . .

Branchlet characters for Juglans nigra, J, cinerea and
progeny of two putative hybrid parent trees . . . . . .

 

A comparison of morphological characters for Juglans
nigra, J, sieboldiana, and progeny of two hybrid trees .

 

Paired affinity index for several Juglans chromatograms

Comparison of paired affinity index with and without
pooling of similar chromatograms . . . . . . . . . . . .

Diagnostic values for Juglans nigra, J, regia and

J, regia x nigra based on paper chromatograms of foliage
extracts, and hybrid index values based on morphological
data . . . . . . . . . . . . . . . .

 

Correlation between hybrid index and diagnostic values

for Juglans nigra, J, regia, and J, regia x nigra hybrids

Relative height and tree form traits of seedlings from
open-pollinated putative hybrid walnut trees . . . . .

iv

Page

15

21

24

26

29

33

35

46

47

50

52

55

TABLE Page
A-l. Morphological characteristics of all Juglans species . . 67

A—la. Juglans seed trees and clones used as sources of

experimental walnut seedlings and grafted trees . . . . 69
A—2. Juglans nigra morphological data . . . . . . . . . . . . 75
A-3. Juglans regia_morphological data . . . . . . . . . . . . 78
A-4. Juglans regia_x nig£a_morphological data . . . . . . . . 80

A-S. Weighted hybrid index (h. i. ) values for seedling

families of Juglans regia, .nigra and J, regia x
nigra hybrids . . . . . . . . . . . . . . . . 82

A—6. Morphological data and weighted hybrid index for
Juglans sieboldiana and J, cinerea . . . . . . . . . . . 85

A-7. Juglans sieboldiana x cinerea morphological data for
traits selected for inclusion in hybrid index . . . . . 87

A—8. Position and description of spots on paper chromatograms
of leaf extracts of Juglans regia, J, nigra, and
putative J, regia x nigra hybrids . . . . . . . . . . . 88

A—9. Relative frequency of occurrence of 79 spots on paper
chromatograms prepared from leaf extracts of Juglans
regia, J. nigra, and J, regia x nigra hybrids . . . . . 94

A—lO. Spot frequency difference (s.f.d.) values for 79 spots
found on paper chromatograms prepared from leaf

extracts of Juglans nigr . regia, and J, regia x
nigra hybrids . . . . . . . . . . . . . . . . . . 98

LIST OF FIGURES

FIGURE Page

1. Weighted hybrid index distribution for seedlings and
ramets of Juglans regia, J, nigra, and putative
J, regia x nigra hybrids . . . . . . . . . . . . . . . . . 20

2. Range and mean of hybrid index values for Juglans
sieboldiana, J, cinerea, and three generations of
putative J, sieboldiana x cinerea hybrids . . . . . . . . 28

 

 

3. Applying concentrated foliage extract at x produced
the best chromatograms . . . . . . . . . . . . . . . . . . 37

4. Composite chromatogram of butyl alcohol-soluble
compounds in Juglans regia foliage . . . . . . . . . . . . 42

 

5. Composite chromatogram of butyl alcohol—soluble
compounds in Juglans nigra foliage . . . . . . . . . . . . 43

 

6. Composite chromatogram of butyl alcohol-soluble
compounds in Juglans regia x nigra foliage . . . . . . . . 44

 

Vi

CHAPTER I

HYBRID WALNUTS AND HYBRID ANALYSIS

 

Over the past century, at least 13 combinations of hybrid
walnuts involving 8 Juglans species have been describedii/ Many of
these hybrids are noted for outstanding growth rate, pest resist-
ance, or other desirable traits (Table 1).

Despite the continuing interest in interspecies walnut hybrids,
they are usually difficult to produce artificially (McKay, 1957;
Shchepot'ev, 1960); the 'Paradox' hybrid (J, hindsii x £2512) intro-
duced by Burbank (Howard, 1945) is perhaps the only walnut hybrid
in commercial production. This hybrid is usually produced by
collecting seed from the Hinds walnut, J, hindsii, growing near a
Persian walnut (J, regia) pollen source; it is also possible to
propagate 'Paradox‘ hybrid clones by trench layering and rooting of
cuttings (Lynn and Hartmann, 1957).

Hybrid walnuts are also difficult to identify in the field
(Manning, 1960; Gervais, 1963). Experienced nurserymen sometimes

recognize walnut hybrids in seedbeds of otherwise pure species, but

 

J] Tree taxonomy throughout the text follows Little (1953) for
native species, and Rehder (1940) for exotics; names of species'
authors are omitted.

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3
the hybrids are infrequent and J, EEEEE.X nigra and J, cinerea x
sieboldiana are probably the only ones encountered with any
regularity.

Thus we have a situation in which the potential value of hybrid
walnuts is acknowledged, but they are hard to produce artificially,
rarely encountered in nature, and difficult to identify with cer—
tainty.

Some of the problems of identification begin with Juglans species
which are themselves variable and sometimes poorly defined. For
instance, J, maigr, the Arizona walnut, has at various times been
treated as a race of J, microcarpa, little walnut, a distinct variety
of little walnut, or a separate species (Sudworth, 1934). Similarly, I
J, hindsii has been considered to be included within J, californica, i
the California walnut (Manning, 1957; Sudworth, 1967), a variety of
the California walnut (Jepson, 1908), or a separate species (Sargent,

1965; and many other authors). Anyone attempting to delimit walnut

species by reference to herbarium collections must recognize that any I
specimen might well have been determined according to species defini-
tions that are no longer accepted.

Only two interspecific hybrids involving J, nigra have been authen—
ticated, those with J, regia and J, hindsii. The hybrid with J, regia
was first described by Carriere in 1863 (Reed, 1937). Several forms
of this hybrid have been named, usually based on nut shape (Rehder,

1940). Black x Persian walnut hybrids are not at all common in nature,

4

however, partly because J, regia_usually flowers about 2 weeks earlier
than J, nigra, and also because of apparent incompatibility. For
instance, McKay (1957) was able to produce only 12 seeds from 5,000
controlled pollinations using Persian walnut pollen on black walnut
pistillate flowers. Some J, regia_x'nig£a_hybrids, such as the vigor-
ous l7-year-old’trees described by wellington (1931), flower profusely
but never bear many seed.

A possible natural intersectional hybrid between J, nigra and J,
cinerea, butternut, has been reported from southern Quebec (Gervais,
.1963), but since the fruit dimensions were well within the range of
black walnut seed size and no other morphological information was
given, it seems prudent to be skeptical about this tree's hybridity.

' Soviet tree breeders have been active in walnut hybridization

for more than 50 years. They have recorded some spectacular successes
that deserve attention and attempts at verification by western breed-
ers. Among these are reports of 46 percent crossability between black

walnut and Manchurian walnut, J, mandshurica, and 16 percent cross—

 

ability between black walnut and mockernut hickory, Carya tomentosa,

 

(Shchepot'ev, 1960). Shchepot'ev (1951) also reported a hybrid between
black walnut and butternut and suggested that the cross was more easily
made if vigorous young black walnut trees were used as the female
parents. Additional information on black walnut hybridization and
genetics has been summarized in a previous paper (Funk, 1970).
Taxonomic analysis of suspected plant hybrids must begin with a

clear definition of possible parent species, including not only the

5
species' taxonomic holotypes, etc., but also representatives of the
normal range of variation. Systematic hybrid analysis of this type
received its first real impetus from field studies by Edgar Anderson,
who developed the well-known hybrid index. As originally outlined by
Anderson (1936) and Riley (1938), morphological characters were
selected which appeared reliable in distinguishing two parent species.
All these characters were defined in a simple, often dichotomous, way
(glabrous vs. pubescent, 2—3 nodes vs. 6-8 nodes, etc.). Conditions
characteristic of one parent species were assigned "0" values while
typical individuals of the other species were usually scored "2";
values for several traits were summed to compute the total index.
Hybrids were expected to have intermediate index values, possessing 0
value for some characters, 2 for others, and l for many.

Anderson's methods proved effective for analysis of many hybrid
populations, especially when combined with graphical representations of
morphological character combinations which he suggested "as a device for
helping the eye to aid the mind." (Anderson, 1957). Nevertheless, he
acknowledged that while ”the pictorialized scatter—diagram methods...
yield quicker and more reliable results than any pure biometrical method
which has yet been devised, they should be considered as temporary
expedients." (Anderson, 1954).

The 'new systematics' that has developed in recent years is usually
based on consideration of biochemical and anatomical as well as morpho-

logical characters and often includes complicated statistical analyses of

6
metric data. Some numerical taxonomists have insisted that a minimum of
50 to 100 characters must be measured in order to assure a reasonably
accurate differentiation of taxa. Nevertheless, there are instances in
which inclusion of irrelevant characters in a hybrid index simply
increases the variation in an otherwise clear set of data. In such
situations, it seems appropriate to omit some characters. Taxonomists
often have found it necessary to assign weights to the several char-
acters that may be included in an index, and Hatheway (1962) preposed the
following logical criterion: "The contribution of a character to an
index should be in proportion to its usefulness in demonstrating a known
or suspected relationship.”

Several types of multivariate analyses have been used in statistical
taxonomic studies, including canonical analysis of correlated traits,
principal component analysis of uncorrelated variables, and discriminant
analysis. As pointed out by Mergen and Furnival (1960), "If one knows
the degree of genetic control of a single character, and if it differs
sufficiently between the parental species, one character is sufficient to
distinguish the hybrid. However, if little information is available on
the variation pattern and its degree of genetic control, ...it is advis—
able to use a combination of several diagnostic characteristics." Dis-
criminant analysis has been used several times to distinguish both
natural and artificial hybrid trees from their parent species. The
analysis indicates the combinations of characters that best discriminate

among the groups of progeny being considered. The principal drawback to

7
all such multivariate analyses is that they virtually necessitate use of
computers.

Another aspect of hybrid analysis treats the progeny of parent trees
that are themselves suspected of being hybrids. One possible objective
is to verify or reject the putative hybridity of the parent through
analysis of the progeny; another objective is to define the progeny as
being Fl-selfed, F2, or backcross hybrids. Ledig, g£_al, (1969) used
discriminant analysis to meet both objectives in a study of hybrid oaks
in North Carolina, and Clifford (1954) has shown that hybridity in

Eucalyptus can be confirmed by analyzing progeny. Seedlings from

 

'intermediate' trees in suspected hybrid swarms of Eucalyptus ggniocalyx

 

and E. elaeophora exhibited markedly greater variance for some morpho-

 

logical traits than progeny of parents 'typical' for the two species.
Presumably the greater variation among the hybrid progeny was due to

presence of F2 and backcross seedlings (Clifford, 1954).

OBJECTIVES

The objectives of this study are to relate morphologic and chemical
characteristics of suspected hybrid walnut seedlings and trees to corre-
sponding characteristics of presumed parent species, and to determine
whether characteristics measured in the hybrids are intermediate, exceed
parental values, or perhaps represent unique hybrid traits or combina-
tions of traits. Information obtained in this study will be pertinent to
walnut tree improvement programs that depend on hybridization to create

additional genetic variation.

CHAPTER II

MORPHOLOGICAL ANALYSES OF WALNUT SEEDLINGS

 

In order to provide a baseline for study of variation within and
between Juglans species, a number of morphological characteristics were
tabulated (Appendix Table A-1) using taxonomic reports (Hegi, 1957;
McMinn and Maino, 1951; Manning, 1952, 1957, and 1960; Ohwi, 1965;
Rehder, 1940; Sargent, 1965; Sudworth, 1934), and my own measurements.
The information summarized in this table has been useful in selecting
traits for further study, especially among those species considered as
possible parents of putative hybrid walnuts. Obviously, such a table can
only indicate the patterns of variation within the several walnut species.
General taxonomic works cannot devote much space to discussion of extreme
intraspecific variation or to suspected—but-unconfirmed hybrids. Refer-
ring to specimens from a number of herbaria may provide adequate geo—
graphic coverage, but it does not ensure that specimens were collected
and prepared in a uniform manner.

Any definitive study of hybrid plants must eventually depend on pro—
ducing a quantity of control—pollinated progeny that can be compared with
their own parents, and through statistical analysis, compared with like

populations of non—hybrid seedlings and putative hybrids of uncertain

9

origin. In the case of tree species, several problems make the task
difficult.

The initial problem is the delay in reaching reproductive maturity.
With any plants, it is convenient to assemble breeding populations in one
or a few nearby areas; it is especially desirable for tree breeding since
the large size of trees necessitates the use of equipment that is cumber—
some to move far. But establishment of breeding arboreta typically
begins with collecting seed, preferably representing a reasonable frac-
tion of the geographic range of the species; seedlings are grown in the
nursery and then transplanted to the arboretum, eventually to flower and
bear fruit. In the case of black walnut, there are occasionally a few
flowers and nuts on 5— or 6-year-old trees, but seed production is not
dependable, even on very good sites, until the trees are at least 10
years old from seed. The Forest Service has a few small plantations
approaching this age, and soon I hope to be able to begin systematic
intraspecific crossing in £3.215E2:

As for other species, since 1968 I have received 107 J, Eggia_and

20 J, sieboldiana seedlots, mostly from indigenous or naturalized stands;

 

43 and 7 seedlots, respectively, germinated well enough to establish at
least small outplantings. Within another 2 or 3 years these trees should
have recovered from transplant shock and grown large enough to be used
for analyses of leaf morphology that would be comparable with those based
on mature trees. Study of flower and fruit characteristics will have to

wait for a few more years. The Forest Service also has a small field

10
plantation of J, major, representing several Arizona locations, but

efforts to establish an assortment of J, microcarpa and J, cinerea geno-

 

types in the field have been frustrated. From 6 J, microcarpa collec-

 

tions, no seedlings survived the winter. In an attempt to augment our

7 J, cinerea seedlings grown from commercial seed of unknown origin, I
obtained several good collections from Iowa and West Virginia; squirrels
destroyed every nut in the nursery. Identification of putative J,

microcarpa x nigra and J, sieboldiana x cinerea hybrids will continue

 

 

to be uncertain until more material can be accumulated.

METHODS

Twenty-three traits of leaf and branchlet morphology were analyzed
in an attempt to determine those that are most suitable for character-
izing Juglans species and distinguishing hybrids. Trees in six planta—
tions in Michigan and Illinois were used for analysis. All trees had
been outplanted for at least one season prior to collecting samples, and
large leaves from exposed portions of the crown and vigorous, fully
mature branchlets were selected for analysis. Herbarium specimens were
pressed and dried and data were tallied as follows:

Leaf length - cm.

Number of leaflets

Longest leaflet:

Position - of pair containing longest leaflet,

counting from tip

ll
Petiolule length — mm.
Blade length — mm.
Blade width - mm.
greatest width
at 1/3 blade length from tip
at 2/3 blade length from tip
Serrations/cm. — beginning at the broadest part of the longest
leaflet
Curvature - greatest deviation from straight line between tip
and base of blade — in mm.

Base shape — average for several leaflets, adjacent to longest

leaflet

l = cordate

3 = truncate

4 = obtuse

5 = right angle

0‘
ll

tapering (less than 90° angle)

Lopsidedness unequal extension of base of leaflet blade along

petiolule - mm.

Pubescence - upper leaf surface ) 0 none

)

— lower leaf surface ) 2

along mid veins only

- rachis 4 = heavier in vein axils
6 = pubescent
8 - tomentose

12

Rachis redness (mature, fresh leaves) - 0 none
1 = faint
3 - reddish pink

5 = maroon

Foliage color (mature, fresh leaves) - l = pale green
3 = "average"
5 = dark green

Branchlet color (Fully mature, current year's shoot growth)

constituent colors: Green Gray Brown
none 0 0 0
light 1 l 1
medium 2 2 2
dark 3 3 3
Branchlet scurf - 0 = none
5 = heavy

Pith color (1— to l l/Z-year-old wood) — 0

very light tan

5 = dark brown
Trichomes at upper edge of previous
year's leaf sear — O = absent
5 = heavy

Data were recorded from more than 500 trees, although not all traits
were determined for each tree. Parent trees and clones are described in

Appendix Table A—la.

13

BLACK WALNUT MORPHOLOGY

Because provenance test plantations and breeding arboreta had
already been established for J, nigra, considerably more data were avail-
able for this species than for other walnuts (Appendix Table Ae2). Note
that these data were collected with the objective of determining mean
values and the variability of several morphological traits within the
species as a whole. Therefore, some plant material was collected from
unreplicated arboretum plantings, and even when taken from adequately
designed test plantations, specimens were often subjectively selected as
being representative, rather than being chosen at random. Thus, the data
are not suitable for rigorous statistical analysis, such as regression on
various climatic and geographic variables that characterize the seed
source. Indeed, it should not be inferred that the tabulated values
accurately represent the areas from which the seed originated, since in
some instances only a single specimen was scored. Therefore, seed source
locations are listed only to show that sample trees came from throughout
the species' natural range, and thus to provide assurance that the over—
all average values given are probably close to the true mean for black
walnut.

In order to determine the sampling intensity that would be necessary
to make adequate estimates of morphological characteristics in sub—
specific J, nigra units (families, clones, etc.), 6 half-sib families
were analyzed. Parent trees are located in Iowa, Illinois, Indiana, and

Ohio; 2 leaves per tree and 3 seedlings per family were analyzed for the

following traits: position of longest leaflet, leaflet index, serration,

leaflet curvature, base shape, and pubescence. Analysis of variance

followed this format:

Source of variation

Families

Trees in families

Leaves in trees

12

18

Expected m.s.

0% + 20% + 30%
2 2

CL + ZOT

017:

Components of variance are shown in Table 2.

The relative contributions of families, individual trees, and

leaves within trees varied considerably, depending on the trait evalu—

ated. The variation related to seed source/parent tree and to seedlings

within families was expected, but it appears that it will be necessary

to analyze perhaps 3 to 6 leaves per tree in order to obtain reliable

mean values for foliar characteristics of individual black walnut trees.

15

Table 2.--Components of variance for selected traits

in Juglans nigra leaves

 

Relative variation due to:

 

: Trees in : Leaves in

 

Trait ; Families : families : trees
Longest leaflet 0.001 0 0.004
Leaflet index 0 0 0.08
Serration 0.33 0.10 1.88
Leaflet curvature 0.07 0.37 3.19
Base shape 0 0.75 0.36

Pubescence 1.01 1.07 2.19

 

 

16

MORPHOLOGY 0F PERSIAN WALNUT AND PERSIAN X BLACK WALNUT HYBRIDS

A few J, regia seedlots representing parents or stands with good
timber form or supposed hardiness to climatic damage produced seed-
lings large enough to be used in morphological analyses. Additionally,
some grafted Persian walnut varieties were planted simply because of
their reported early or late flowering habit. Data for these seedling
families, stand collections or ramets are given in Table A-3.

Morphological data similar to those for J, regia and J, EJg£a_are
given in Table A—4 for putative J, regia_x nJg£a_hybrids. As might be
expected, variation was greater in the hybrids than in either parent

species in nearly all traits for which it was calculated.
COMPARING SPECIES AND HYBRID POPULATIONS

Without actually making a rigid discriminant analysis, I have tried
to achieve the same results by examination of the statistics in Tables
A—2, A—3, and A-4. The objectives were to select those traits in the two
species and their hybrid that differ widely in their mean values, but at
the same time do not vary much within taxa (Kendall and Stuart, 1966).
Based on these requirements, characters such as leaflet length and base
shape were not useful because means for both parent species and the
hybrid were too close to be distinctive. The index of leaflet curvature
values were also similar and furthermore, variability was rather high,
with coefficients of variation ranging from 38 to 54 percent. Other

traits scored, such as leaf length, leaflet lopsidedness, rachis redness,

17
foliage and branchlet color, branchlet scurf, pith color, and leaf scar
trichomes were not subjected to any sort of formal statistical analysis
because of obvious excessive variability within the species (such as leaf
length and coloration), virtual absence from all samples (scar trichomes)
or insufficient data (pith color).

Initially, I thought that branchlet color might be a useful diag-
nostic characteristic since Persian walnut seedlings seemed to have a
notably greenish tinge While black walnuts tended to be more brown, but
seasonal changes hampered effective scoring. I assumed that branchlet
color changes would be completed during the autumn, at least by the time
of leaf fall. But when scoring a J, nigra.plantation, I tallied 2 blocks
in mid-November and 4 blocks in early December. Surprisingly, analysis
of variance showed the effect of 'blocks' to be significant for the green
component of branchlet color. The trees scored in November were 54 per-
cent more green than those rated in December; there was no seasonal
change in the brown or gray components. As recently pointed out by Perry
(1971), hardwood seedlings contain sufficient chlorophyll in their bark
and buds to carry on some photosynthesis throughout the 'dormant' season.
Apparently if differences in branchlet color are to be used in Juglans
taxonomic analysis, any populations to be compared will have to be

scored at the same time and place.

l8

Calculating a Hybrid Index

 

The following five traits of leaf morphology were selected as likely

to be useful in distinguishing Juglans regia and J, nigra seedlings and

 

putative hybrids between these two species:

leaflets per leaf

marginal serrations per centimeter

position of the longest leaflet pair on the leaf

leaflet length:width index

pubescence, combining both leaf surfaces plus the rachis
For each of these traits, J, nigrapseedlings tended to have higher
scores, with J, regia low and hybrids intermediate. They were thus
directly and easily usable in calculating a hybrid index.

Many modifications have been suggested to improve and refine
Anderson's (1936) original concept of the hybrid index which he acknowl-
edged to be crude (Anderson, 1949). Essentially, the proposed changes
involve using metric.data or scales expanded beyond the few values used
by Anderson, incorporating more than a few traits in the index, and
weighting the index, either to 'equalize' the contributions of the
several components, or to adjust their weight in proportion to their
assumed relative usefulness. I have tried to make all three kinds of
improvements in deriving an index useful for differentiating Persian x
black walnut hybrids. As previously described, I used measurement or
count values, or at least a 5-point scale to score each trait, and

selected 5 traits to make up the index. Rather than select some

l9

arbitrary basis, it seemed logical to weight each trait according to its
relative constancy within the parent species; that is, inversely to its
relative variability. Several statistics describing sample variation
might well have been used, such as the standard deviation, variance, and
standard error of the mean. I chose standard error of the mean for
weighting since it tends to minimize differences in sample size and
should be equally reliable whether used in indexes based on family or
clonal means or in those representing individual trees. Weighting values
were derived as shown in Table 3, followed by the computation of a hybrid
index for the overall average of J, £2512 and J, 2Jg£a_as examples.

Weighted hybrid indexes were calculated for J, regia half-sib seed-
ling families and grafted ramets, J, nigra seedling families, and an

assortment of suspected J, regia x nigra hybrids. Individual seedling

 

and ramet index values are shown in Figure l, and family mean indexes as
well as those for individual trees are given in Table A—5. The index
does a good job of separating Persian from black walnuts and the distri-

bution for both species appears to be reasonably normal.

Separation of Hybrids from Parent Species

 

The putative hybrids show considerable overlap of both of the pre-
sumed parent species. With only a couple of exceptions, those designated
as F1 ramets were provided by Dr. John W. McKay from the Agricultural
Research Service walnut collection at Beltsville, Maryland; four of them

were produced by control-pollination, and I am inclined to take his word

 

20

.mvauph: mmmwm x mmmmw .fl u>auwu=n can .mmmmm am .MNMMH mmmmmmw mo muuanu was mmcaavuou you coausaauumav xovaa vamp»: vou:w«u3ll.a unawam

xouca saunm:

con owN ooN o¢~

    

 

mafia m>mos
casua: «yuan .m

 

 

spawns: coauuuonom vooca>vu I

scares; am . wwwwwm

 

oaaa
m>mon casuau
mwmou .m

 

 

on on

0H

ma

hi
(\I

X3P“I PIJun

 

21

Table 3.-—Statistics used in computation of hybrid index for

 

Juglans regia and J, nigra

 

Leaflets : Serrations : Longest : Leaflet

per leaf : per cm :leaflet position: index : Pubescence
:regia :nigra:regia :nigra: regia : nigra :regia :nigrazregia: nigra
8.72 18.36 0.77 4.88 .135 .218 2.13 2.69 0.49 10.29

0.14 0.51 0.14 0.17 .008 .009 .032 .031 0.04 0.30

0.33 0.16 0.009 0.032 0.17

 

8.72/0.33 + ...0.49/0.l7

Hybrid index value for

average J, regia 26.42 + ...2.88

115.67

18.36/0.33 + ...10.29/0.l7

Hybrid index value for

average J, nigra 55.64 + ...60.43

254.95

22

for the authenticity of the remainder. But Dr. McKay expressed doubts
about the parentage of two of the hybrids, including our number NC—5959,
the Fort Hunter walnut. This tree has a hybrid index of 215, greater
than that for three of the black walnut trees sampled, and I suggest that
it may be a backcross to black walnut. The other hybrid that McKay
questioned is NC—5954, the Hillgate walnut; an index of 159 puts it
squarely in the middle of the other hybrids.

The four Fl hybrids with index values that oVerlap the range of
Persian walnut (index between 130 and 140) include one of McKay's pedi—

greed J, regia x nigra crosses (NC—5924), so we cannot confidently state

 

that a walnut with a hybrid index in the 130's is "nOt an F1 hybrid."

The 60 trees designated as advanced generation hybrids are progeny
of open-pollinated Fl's. Such trees are usually referred to as Fz's, but
the designation is not necessarily correct since the parent trees may
have been self—pollinated, backcross-pollinated by a tree of one of the
parent species, or conceivably outcrossed to a third Juglans species.

The three hybrids with the highest indexes (250+) all came from
Beltsville where there was great opportunity for the hybrid parent

trees to backcross with black walnuts. Four of the six hybrids with an
index of less than 110 came from a hybrid tree owned by a nut grower in
Indiana. It is easy to imagine that this tree backcrossed to his Persian
walnuts, perhaps to a single cultivar that shed pollen at the appropriate
time.

It is tempting to.suggest that the large group of advanced genera—

tion hybrids with indexes between 120 and 150 may be comprised of Fz's.

 

23
This suggestion is purely speculative but seems not illogical, especially
considering the arrangement of the hybrid walnut plantations at Belts—
ville in which grafted ramets of many Fl's were planted in adjacent rows
and could easily intercross. At any rate, analysis of the progeny tended"
to confirm the hybridity of the parents (presumed to be Fl's), as shown,
for instance, by greater variation within hybrid half-sib families than
is found in the intraspecific half-sib families (Table 4). This simple
table conforms to the pattern of increased phenotypic variance that would
be expected if the hybrid families represent segregation among an
increased number of genotypes (Clifford, 1954). For each size of family,
the mean range between lowest and highest hybrid index is greater for
seedlings of hybrid parentage than for seedlings of either single species.
Another indication of parental hybridity is poor germination; seedlots
from 25 J, regia_x nigra parent trees averaged only 32 percent in three
nursery tests. This low germination is not surprising, considering the
reported infertility of Persian x black walnut hybrids (McKay and McKay,
1941); it is well below average germination values reported for black
walnut, which range from 60 to 75 percent (Newbold, 1967; U.S.D.A.,

1948).

 

24
Table 4.--Range of hybrid index within half—sib walnut families;
table values show the difference between high and low

hybrid index in each family

 

 

Taxon of : Number of seedlings per family
Jparent tree : 2 3 4 5 6 8
Juglans regia 28 29 26 26 42
13 25 19 32 25
4 31 52
ll
__2____
Mean range 15 21 23 29 40
J, nigra 12 28 22 54
26 4O 50
25 58 46
1 13 33
l 39
12 26
11
7
l
_3___ _
Mean range 10 28 33 41
J, regia x nigra 39 56 56 59 44
35 20
98

Mean range 37 56 58 59 44

 

 

25

BUTTERNUT AND JAPANESE WALNUT MORPHOLOGY

As previously mentioned, in the Forest Service research plantations
in southern Illinois there are only a few small trees of Juglans

cinerea, butternut, and J, sieboldiana, Japanese walnut. There are also

 

a couple of grafted heartnut QJ. sieboldiana cordiformis) varieties, and

 

I collected foliage from several trees in Michigan in an attempt to
characterize the two species. Morphological data are summarized in
Appendix Table Ar6.

Even though the data were limited, butternut and Japanese walnut
could be distinguished by several morphological features, but not the
same ones used to differentiate Persian and black walnuts. Such traits
as number of leaflets, position of the longest leaflet, serrations per
cm, and leaflet index were quite similar for J, cinerea and J,
sieboldiana; others, such as leaf length and leaflet curvature appeared
to differ between the two species, but these data varied too widely to
be reliable. I selected leaflet length (column 7 in Table A-6) and
relative lopsidedness (l4), rachis pubescence (15c), and pith color (16)
as being most useful for distinguishing butternut and Japanese walnut
and calculated a weighted hybrid index using the statistics in Table 5.
Hybrid index values for the average of the two species are also shown.
Most traits had lower values for Japanese walnut than for butternut,
but since pith color scores were higher in Japanese walnut, they were

subtracted when the indexes were calculated.

 

26
Table 5.-—Statistics used in computation of hybrid index

for Juglans sieboldiana and J, cinerea

 

 

: : Rachis
: Leaflet length : Lopsidedness : ,pubescence : Pith color

 

sieb. : cin. : sieb. : cin. : sieb. : cin. : sieb. : cin.

 

NI

Avg.

145.6 101.3 .020 .008 6.6 3.5 0.85 2.9
9.7 4.0 .003 .001 0.80 0.23 0.13 0.82
S- 6.9 .002 0.52 0.48

 

Hybrid index value for 145.6/6.9 + .020/.002 + 6.6/.52

average J, sieboldiana - 0.85/0.48

 

21.1 + 10.0 + 12.69 - 1.77

42.02

101.3/6.9 + .008/.002 + 3.5/0.52

Hybrid index value for
average J, cinerea — 2.9/0.48

14.68 + 4.0 + 6.7 - 6.04

19.37

 

27

BUTTERNUT X JAPANESE WALNUT HYBRIDS

Foliage was collected from several putative F1 hybrids as well as
from their open—pollinated (F2?) seedlings, and from advanced-generation
(F3?) progeny of the seedlings. According to the owner of the F1 parent
trees NC—5827 through NC—5831, these trees are themselves half—sibs;
interpollination among them must result in some inbreeding. Data repre-
senting the traits selected for inclusion in the weighted hybrid index
are given in Appendix Table A—7 along with h.i. values for each collec—
tion.

In the five lines for which I have data from both parent trees and
progeny, h.i. was reduced in each successive generation, but as shown in
Figure 2, the index values for each generation group overlapped consider-
ably. Furthermore, the index values for parents and progeny are not
correlated (r = -0.17). My small sample of 34 seedlings may include more
than the expected proportion of extreme segregants, and of course, hybrid
index as such is not a heritable characteristic, but since the index is
based on real morphological features, it seemed reasonable to expect a
stronger parent-progeny correlation than was shown.

One possibility is that a tree with a low h.i., such as NC—5830,
might have served as pollen parent for many of the seedlings. I visited
the trees during flowering season and estimated the probable pollinator
for each of the trees; my conclusions are shown in Table 6. Plainly, I
picked the 'wrong tree' as the probable pollinator to verify my hypothe-

sis. Indeed, the estimated and actual h.i. values for the progeny had a

 

28

 

.mvaunan mouoawu x mamavaonoam am.o>aumusa

 

mo maoauouocow counu can .mouoaao am .mcoavaopoam mandmsh you mosam> xov:« wanna: mo coca was ow:mmnu.~ oasmam

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

\o
I ca
1 ON 1
II, M
m
1...! II II 0.
1
I.
II [I II P
Ll II 1 omfl _
P 1
«0.3.36 .w I] m _
.1 cc
all
mucouma Hm
I. on
Acowoum mm zcowoua mm mamavaonoam am
.11

co

29
Table 6.-—Hybrid index comparisons for hybrid walnut seedling

families and assumed parental combinations

 

Progeny H.I.
Assumed : Estimated :
Female parent : male parent : from
NC No. : H.I. : NC No. : H.I. : ,parents : Actual

 

 

5827 35.2 5828 40.5 37.8 28.6

5828 40.5 5828 40.5 40.5 25.0

5829 31.9 5827 and 37.8 34.8 30.6
5828

5830 27.4 5827 and 37.8 32.6 25.4
5828

5831 29.0 5828 40.5 34.7 27.6

 

 

30
correlation coefficient of —O.21, worse than before.

I believe that the five hybrid trees in Michigan were not only
intercrossing and self-pollinating but also to some extent backcrossing
to local butternuts. Since the average index of the presumed F1 hybrids
was 32.8, while their open-pollinated progeny averaged 27.5, and as
previously calculated, the h.i. for average butternut was 19.4, a simple
equation can be solved to estimate the proportion of butternut pollen
introduced into the 'F2' generation.

Let x = the proportion of butternut pollen

Then 19.4x + 32.8 (l-x) 27.5

.395

and x
Assuming that intercrossing among the five parent hybrids 'averaged
out', I conclude that almost 40 percent of the seedlings are butternut—
backcross progeny.
Finally, the low h.i. values for all five trees in family NC—5826
(Figure 2) which averaged only 15.7, lead me to believe that the parent

tree was not a hybrid at all, put a pure butternut.

 

31

BLACK WALNUT HYBRIDS WITH OTHER SPECIES

The walnut hybrids described so far may be considered as intra-

sectional hybrids, with Juglans regia and J, nigra being in Dode's (1906,

 

1909) section Rhysocaryon, and.J, cinerea and J, sieboldiana in section

 

Cardiocaryon. Dode did in fact place J, cinerea in a separate section,
Trachyocaryon, but he is a noted taxonomic 'splitter', and Manning
(1957) "does not consider [Trachyocaryon] distinct from Dode's section

Cardiocaryon of Asia, an opinion shared by Nagel, 1914...."

Black Walnut x Butternut Hybrids

 

A natural intersectional cross between J, nig£a_and J, cinerea is
certainly possible since the two species have overlapping ranges through
a large area extending from Minnesota and Missouri to New York and North
Carolina. But as discussed by Wright (1962), "as a general rule species
which occupy the same sites in the same region do not cross with each
other. Otherwise, how would the species distinction have been main-
tained?" This rule seems to hold for black walnut and butternut; even
though the two species are morphologically and ecologically similar,
there are no published accounts of naturally occurring hybrid swarms,
and as previously mentioned, only a few references to artificially pro-
duced hybrids.

Considering this situation, I was skeptical about the actual
hybridity of two lots of seed from putative J, 21g£a_x cinerea hybrids

in Cass County, Michigan. I searched for the parent trees in 1970 and

 

32
found that NC-5801 (= MSFG 848) had apparently been cut. At the loca-
tion given for NC-5802, I could find no trees that I would consider to
be other than ordinary black walnuts, but could not be certain that I had
really located tree NC-5802. I imagine that the parent trees were sus—
pected as hybrids primarily because they produced rather elongate nuts.

As can be seen in Tables A—2 and Ae6 and in most dendrological keys,
leaf characters of black walnut and butternut do not differ sufficiently
to distinguish the two species. Instead, they are usually differentiated
on the basis of flower morphology, fruit and nut texture, mature bark
pattern, pith color, and presence of the 'moustache' of trichomes above
the previous year's leaf scar. In our young plantations, only the latter
two branchlet characters could be used in taxonomic analysis; they are
the same two used in Harlow's (1948) Twig Key. In Table 7, average
values for these two characters are compared for black walnut, butternut,
and the NC-5801 and NC-5802 seedlings.

For both pith color and the presence of leaf scar trichomes, the
NC—5801 seedlings scored even lower than the average values for black
walnut. I suggest that the parent tree was pure J, nigra, but certainly
an interesting seed tree as discussed under progeny test results in
Chapter IV. NC-5802 can still be classed as a putative hybrid. Although
pith color was as dark as or darker than that of the butternut collec-
tions I sampled, the average value for leaf scar trichomes was higher
than any recorded for black walnut, but lower than any of the butternuts.

This seedling family deserves further study, but one intermediate

33

Table 7.--Branchlet characters for Juglans nigra, J, cinerea,

 

and progeny of two putative hybrid parent trees

 

 

 

: Sample : Pith color : Leaf scar trichomes
Taxon size : x : Snif r : i.
J, nigra overall 1.45 0.27 0.04 0.01
average
J, cinerea ” 2.93 0.82 3.50 0.49
NC-5801 31 0.95 0.11 O --
NC-5802 10 3.65 0.18 1.25 0.13

 

J] Standard error of seedlot mean.

 

34
characteristic is hardly sufficient to make a case, and positive
classification must be deferred until the trees have flowered and

fruited.

Black Walnut x Heartnut Hybrids

 

Our plantations include seedlings from two trees designated as J,

nigra x sieboldiana cordiformis. The parent tree for seedlot NC—5845

 

was apparently a grafted ramet and the nuts were labelled as having been
collected from the hybrid variety 'Leslie Burt'. Only two seedlings
survived from seedlot NC-5848, collected from 'a second-generation
hybrid'. Selected data from Tables A-2, A—6, and the two hybrid seed-
lots are assembled in Table 8. The tendency for the progeny to resemble
black walnut in some traits, Japanese walnut in others, and to be inter-
mediate in about one—fourth of the statistics, provides good verifica-
tion for the hybridity of both parents. As shown in Chapter IV, NC-5845
seedlings were also characterized by the combination of good form and

above average growth rate in Illinois test plantations.

 

35

canoe uoavwmm mo Houum wuva6um \fl

 

 

m to m.o ~.m m.o o.o ~.o m.m moamommnam magnum
N .0 s.o o.o o.o o.m ao.o so.o masoeoauu “mom mama
m.m-a o.o a.~ a.o m.o a.o m.a uoaou euam
m.mnm «.0 s.~ m.o m.~ a.o. m.m mamas «mam
m.~nm m.o e.m m.o m.m N.o m.e an “an maoaumuuom
oaauww m.m maa m.m sea o.m maa aaav euwama soaamma
ma um m.o m.ma o.a m.ea m.o m.ma A.oav mumaamua
owamu \AMm m \wmm m \flmm m uaumauMum
N m coma Hamuo>o coma Hamuo>o ouwm mamamm
wcwmtoz mqwmloz I. :oon

 

.eaonoam .n mamas mm

0. O.
O. O.

 

 

.aaaaeaonoau .s

moouu pawn»: can mo msowoua was

.oumaa maoamam.uow.mu0uoouono Hmofiwoaonmuoa mo comauomaoo <Ilow manna

 

CHAPTER III

CHROMATOGRAPHIC ANALYSES 0F WALNUT FOLIAGE EXTRACTS

 

Paper chromatography has proved to be a useful adjunct to morpho-
logical analysis in several taxonomic studies (Carter and Brehm, 1969;
Dugle, 1966; Hunter, 1967; McHale and Alston, 1964), and in some studies
of plant hybridization, chemical analysis has successfully been used
alone (Alston and Hempel, 1964; Alston and Simmons, 1962; Hanover and
Wilkinson, 1970; Williams, 1955). In some cases, species-specific com—
pounds representing both parental species were found to accumulate in the
hybrids (Alston, g£_gJ,, 1962), while with other species, unique com-
pounds appeared in the hybrid that were not present in either parent
(Alston, gt ai., 1965).

Paper chromatography was used to analyze Juglans foliage extracts
for presence of polyphenols following the methods of Hanover and Hoff
(1966). More than 90 chromatograms were developed, using both fresh and
dry leaves as the starting material, preparing extracts of several
different volumes and concentrations, and using several combinations of
spot position and solvent flow direction on the chromatography paper.

In preliminary analyses, fresh and dried leaves both generated satis-
factory extracts, using 20 gm and 10 gm samples, respectively. Diluting
the concentrated butyl alcohol fraction to only 1 ml sometimes yielded

36

 

37
an extract so viscous that when it was applied to the paper the spot
failed to dry. Using more alcohol to dilute such heavy extracts to
1 1/2 to 2 ml produced solutions and spots that dried promptly. Even
so, several chromatograms were excessively streaked. A series of tests
using aliquots of between 20 and 50 pl of the concentrated extract,
indicated that 20 or 25 ul was sufficient to produce a chromatogram
that exposed a large number of compounds and one in which streaking was
not severe enough to prevent distinguishing most spots. In the course
of these repeated trials, the concentrated extracts of the butyl alcohol
fraction yielded good chromatograms even after cold storage of the
extract for 8 months or more.

Finally, I compared the quality of papers produced by spotting in
all 8 possible positions (each of 4 corners, front and back), and also by
running the first—direction solvent across the short dimension of the
paper and the second solvent the long way rather than the conventional
long—way-first procedure. Spotting the extract on the upper side of the
paper in the upper-right-hand corner (Figure 3) produced the best chro-

matogram.

 

<

paper machine
direction

 

 

 

Figure 3.--Applying concentrated foliage extract at "x"

produced the best chromatograms.

38
Running the first-direction solvent across the short dimension of
the paper produced no better chromatograms, but different compounds were
occasionally exposed. Among other idiosyncracies of the technique, I
finally had to accept the fact that papers placed in the front rack of
the cabinet would not make good chromatograms; putting a blank paper in

the front did seem to improve the quality of the four papers behind it.

   
 

39

CHROMATOGRAPHIC TECHNIQUE

The following technique produced the best spot separation and

definition.

1.

10.

Collect 20 gm fresh leaves or 10 gm air—dry leaves

from herbarium specimens. Remove rachis and coarse
portions of petiolules before weighing.

Extract for 2 minutes in 100 ml boiling water acidified
to 0.05 percent HCl.

Macerate in semi-micro blender for 1 minute at high
speed.

Suction-filter through Buchner funnel containing Whatman
No. 4 filter paper plus l/4-inch pad made of filter paper
macerated in water.

Return macerated leaves, pad, and filter paper to beaker,
add 100 m1 boiling water and boil 2 minutes more.
Macerate as in 3.

Refilter as in 4.

Wash macerated leaves and filter paper with 50 ml boiling
water and filter to dryness. Discard solid residue.
Allow filtrate to cool in separatory funnel. Gently
wash this water extract 5 times with 50 ml ethyl ether

to remove waxes and fats. Discard ether extract.

Wash remaining water extract 5 times with 50 ml normal
butyl alcohol. Remove emulsion by decanting and by cen-

trifuging at 4000 r.p.m.

ll.

12.

13.

14.

15.

16.

4O
Reduce butanol fraction in rotary film evaporator at
50—55° C. When virtually dry, take up in 1 ml n butyl
alcohol.
Spot Whatman 3MM chromatography paper in the upper right
hand corner as it comes from the box with 25 ul of con—
centrated extract and place in cabinet for descending
chromatography.
Enter first-direction solvent, made in proportions of 4
butyl alcohol: 1 glacial acetic acid: 5 water. Solvent
must be mixed in advance and allowed to stand in separatory
funnel all day; the water/acetic acid (lower) layer is then
discarded and a few drops of butyl alcohol added to mini-
mize dissolution. Develop first—direction down the long
dimension of the paper in the same direction as is shown
on the box as "machine direction”. A typical run requires
15 to 19 hours to descend to bottom of paper.
Remove papers, dry them in fume hood and return them to
cabinet.
Enter second-direction solvent-—10 sodium formate: 200
water: 1 formic acid. Typical second—direction run
requires 4 to 6 hours.
Remove papers and dry as before. Outline spots and label
as to color shade under visible and long-wave ultraviolet

light.

 

41
17. Expose papers to ammonia fumes for one hour. Outline
spots and label as before under both visible and ultra-
violet light.
18. Spray papers with freshly prepared diazotized sulfanilic
reagent; outline and label spots.
19. Spray promptly with 2N sodium hydroxide; outline and

label spots.

PAPER CHROMATOGRAPHY AS AN AID IN IDENTIFYING

PERSIAN X BLACK WALNUT HYBRIDS

Paper chromatograms prepared from 32 foliage samples proved to be

usable in the attempt to distinguish Juglans regia, J, nigra, and puta-

 

tive hybrids between the two species. In the several chromatograms, a
total of 79 different spots was distinguished. The average position of
these spots on the paper chromatograms is shown in Figures 4, 5, and 6,
and their characteristic color under visible and ultraviolet light, fol-
lowing exposure to ammonia fumes and after two reagent sprays is given
in Table A—8. I made no attempt to identify the compOunds that were
represented by co-chromatography with known pure chemicals, elution of
the spots followed by spectral analysis, or any other method. However,
I do speculate that spot 10 which was present on every paper may be

hydrojuglone glucoside or a related juglone derivative.2/

 

2] Hess, Charles E. The vegetative propagation of black walnut
(Juglans nigra L.). A report of research conducted under Cooperative
Agreement Supplement No. 16 to the Master Memorandum of Understanding of
June 1, 1953, between the Forest Service and the Purdue Agricultural
Experiment Station, Contract No. A9fs—12234, 11 pp.

 

 

Solvent Front

42

‘— Butyl alcohol: acetic acid: vatar

mm

 

 

UV

a

 
 

,0
.“9o

Origin

*— Joan :ppa am; :oamo; mxpos

 

 

Solvent Front
Figaro 4.-—Conpoaitc chromatogram of butyl alcohol aolubla compounds in Juglana regia foliage.

 

'~—‘ ‘_M“W"' ,, "‘ r ’7’-

Solvant Front

43

+—— Butyl alcohol: acetic acid: water

 

 

 

 

 

Sol vent Front

Figure 5.--Conpoaito chromatogram of butyl alcohol aolubla compounds in Juglanfi nigra foliage.

Jaaen :ptoo owe; :ozamo; unypog

+—

44

 

in .u.iana regia a nigra foliaga.

 

conpounua

 

hol soluble

urc 6.--Conpoaite chrolatcgran o‘ Lutvr ciao

 

 

 

 

45

Paired Affinity Index

 

I calculated paired affinity (PA) indexes for several pairs of
chromatograms (Table 9). The PA index was designed "to compare pairs of
species in terms of chromatographic affinity (or presumptive biochemical
affinity)" (Ellison, g£_aJ,, 1962), and I assumed that it might be suit-
able for individual chromatograms. The index was calculated as follows:

PA = spots common to A & B x 100
total spots in A + B

 

There seemed to be an upper limit to PA values of about .65 to .70
using my technique, with interspecific comparisons expectably lower, in
the .35 areaa For instance, two chromatograms spotted with the same
extract must 'contain' the same compounds; but the analytical technique
was not adequate to expose all of them. Such discrepancies may be at
least partially explained by differences in environment within the
developing cabinet and by the fact that some spots were simply overlooked
when the chromatogram was being scanned. The relatively low correspond-
ence between papers run with two samples of the same extract suggested
that a composite value might be appropriate. Thus, when chromatograms
70-1, 70-9, and 70—10 were 'pooled' (they were all derived from the same
tree), they had a total of 62 spots. Similarly, 70—17 and 70—17—S
together had 33 spots, and 70-18 and 70-18-S had 36. When PA indexes
were calculated for these pooled chromatograms, the values tended to be
somewhat higher as shown in Table 10. Pooling apparently allowed a
closer approach to exposing all the polyphenols present in an extract,

and should minimize 'differences' between two chromatograms that are due

46

Table 9.--Paired affinity index for several Juglans chromatograms

 

 

 

 

Chromatograms : Spots
compared :Common:Total: PA : Remarks

70-1 : 70-9 37 59 .62 Dry leaves vs. fresh leaves,
70—1 : 70—10 39 6O .65 same population
70-9 : 70-10 35 54 .65 Two papers from one extract
70-17 : 70—17-S 23 33 .70 Changed paper orientation
70-18 : 70—18-S 24 36 .67 Changed paper orientation
70—10 : 70-15 26 50 .52 Comparisons among 5 Juglans nigra
70—10 : 70-16 27 48 .58 collections (10 pairs of chro-
70—10 : 70-17 26 49 .53 matograms)
70-10 : 70-18 22 50 .44
70—15 : 70—16 23 39 .59
70-15 : 70-17 25 37 .68
70—15 : 70—18 22 37 .59
70-16 : 70-17 25 36 .69
70-16 : 70—18 17 41 .41
70-17 : 70—18 17 4O .43

7b : 8b 23 46 .50 Two J, regia_samples

7b : 70-10 24 56 .43 J, regia_: J, nig£a_comparisons

7b : 70—17 16 49 .33 (4 pairs of chromatograms)

8b : 70—10 21 57 .37

8b : 70—17 16 47 .34

 

47
Table 10.--Comparison of paired affinity index with and without

pooling of similar chromatograms

 

 

 

Pooled chromatograms : Spots :Pooled:Regular:Single chromato—
compared :Common:Total: PA : PA : grams compared
70-1, -9, -1O : 70—17, -l7-S 33 63 .52 .53 70-10 : 70-17
70-1, -9, -10 : 70—18, -18-S 36 62 .58 .44 70—10 : 70-18

70—17, —l7-S : 70-18, —18-S 27 42 .64 .43 70—17 : 70-18

 

48
to weak or overlooked spots rather than truly distinctive compounds.
Whenever possible, two chromatograms should be prepared from each
extract and a composite table of RF values prepared. Even by follow-
ing this precaution, I doubt that the PA index can be used satisfactorily
to assess the hybridity of individual suspect trees; the technique seems
more appropriate for classification of populations such as seedling

families or hybrid swarms.

 

Diagnostic Values

Some spots, such as l and 10, were found on all papers for both
Persian walnut, black walnut, and their hybrid, and therefore could not
be used to distinguish the taxa for this study. Others, such as 50 and
54, were found in all black walnut chromatograms and those for most of
the hybrids, but never in the Persian walnut papers, and might be classi-
fied as good indicators of 'non-rggiaf trees. Other spots tended to be
more prevalent in one taxon than in the others, but the distinction was
not usually absolute.

In order to quantify these differences, I developed 'diagnostic
values' based on the simple presence or absence of the 79 spots on each
chromatogram. The values were computed from a table of decimal fractions
indicating the relative frequency of occurrence of the compounds in all
the chromatograms for each species or the hybrid (Table A—9). These
decimal fractions, designated 's.f.' for spot frequency, were used to
calculate spot frequency differences (s.f.d.) for each spot as being

more characteristic of J, nigra (s.f.d. relatively high), J, regia

49

(s.f.d. relatively low), or J, regia x nigra hybrids (intermediate).

 

To obtain the appropriate s.f.d. values when comparing two taxa, the
calculations were made by subtracting spot frequencies for hybrids from
those for J, nigra; similarly, J, regia_spot frequencies were subtracted
from hybrid frequencies and J, regia_fractions were subtracted from J.
nigra, Thus, to calculate the s.f.d. values given for spot 2 in Table

A-lO, spot frequencies were taken from Table A—9 as shown below:

nigra - regia: .53 - .22 = .31

nigra — hybrid: .53 - .38 = .15
hybrid — regia: .38 — .22 = .16

Spot frequency differences for each spot that was present were
summed to arrive at the diagnostic value (d.v.) for one chromatogram.
Thus, the diagnostic value indicating that chromatogram 70-1 represents
a black rather than a Persian walnut was calculated as follows:

d = s.f.d. + s.f.d. + s.f.d. ... + s.f.d.

'V°7o—1 l 3 4 83
11.6 o + .25 - .33 ... + .47

Note that s.f.d.'s for spots 2, 12, 14, etc., are not included in the
calculation since as shown in Table A-9, these spots were not identified
on paper 70-1.

Diagnostic values for 32 chromatograms are given in Table 11 along
with hybrid index values in each instance that hybrid index was calcu-
lated for the same material. Spot frequency differences were initially
calculated on the basis of relative frequency of the compounds in chro-
matograms representing black and Persian walnuts. Diagnostic values

computed from these spot frequency differences separated the two species

50
Table ll.--Diagnostic values for Juglans nigra, J, regia, and J, EggJa_x
nig£a_based on paper chromatograms of foliage extracts, and hybrid
index values based on morphological data.

(Hybrid index values from Table A-5 are given for comparison)

 

 

 

Chromatogram :Accession: _7 Diagnostic values .1, : Hybrid
number : number : E} VS° E: : 2: VS' h. : h- VS° £3 : index
J, nigra
70- l 6156 11.6 9.1
- 9 6156 12.1 8.4
-10 6156 11.1 7.5
—15 6137 9.9 7.4
-16 6160 8.4 5.4 268
-17 6163 9.3 4.5 254
-18 6175 9.0 4.2
-19 6166—4 9.0 6.6 257
-20a 6163-6 9.0 7.4 253
-20b 6163-6 10.3 7.8 253
-22 6159-4 7.6 6.9 258
-23 6163-9 13.3 9.3 245
-24 6138—5 11.7 8.4 264
—25 6160-10 9.1 5.7 280
—26 6138-7 9.7 9.3 261
J, regia
7b -l.6 -5.0
8b —0.9 -5.9
71- l 5892 —1.2 —3.8 124
- 2 5899 -O.9 -3.5 119
— 3 5902 -3.4 -3.9 119
- 5 5907 -4.9 -5.6 126
- 6 5908 -5.8 -5.8 111
- 7 5909 -4.7 -6.1 113
- 8 6196 -3.8 -6.2
J, regia x nigra
71- 9 5881 8.4 -4.8 13.2 187
-10 5927 9.0 -4.5 13.4 165
—11 5954 6.1 —5.2 11.8 159
-12 5959 7.9 —4.6 11.7 215
-13 5846 0.8 -5.6 6.4 128
-14 5880 1.3 -3.6 4.9 159
-15 5919 3.1 -5.2 6.2
-16 5928 3.9 -5.6 8.9 159

 

51
quite well, with J, nJg£§_diagnostic values ranging from 7.6 to 13.3,
and values for J, regia_between -0.9 and —5.8. When the nigra vs.
£§g1a_spot frequency differences were applied to hybrid walnut chro—
matograms, they tended to have nicely intermediate diagnostic values,
but the range from 0.8 to 9.0 included three values overlapping the
range for black walnut; one of them was the questionable Fort Hunter
hybrid, NC-5959. The diagnostic value of 7.9 calculated for this clone
adds support to the prospect that this hybrid is a backcross to black
walnut. The Hillgate hybrid (NC-5954) was once again satisfactorily
separated from either putative parent species with a diagnostic value
of 6.1.

Since the diagnostic values calculated from.nig£a_vs. regia spot
frequency differences did not completely separate the hybrids from the
two species, I also calculated diagnostic values based on nig£a_vs.
hybrid and.£ggia_vs. hybrid differences. These species vs. hybrid
values fell into two quite distinctive groups (Table 11).

As might be expected, the diagnostic values based on paper chromato-
grams were correlated with the hybrid index values based on leaf mor-
phology. Correlation coefficients are given in Table 12 and show that
hybrid index was highly correlated with overall diagnostic values, no
matter which pair of taxa was used to compute spot frequency differences.
When the hybrid index:diagnostic value correlation was made for the
hybrids only, the diagnostic values based on nig£a_vs. £2513 spot fre—
quency differences were best correlated with hybrid index, even though

these values were the ones that showed some hybrid clones overlapping

52
Table 12.--Correlation between hybrid index and diagnostic values

for Juglans nigra, J, regia, and J, regia x nigra hybrids

 

 

 

 

 

Taxa compared to : Correlation coefficients
compute spot : based on:
frequency differences : All samples : Hybrids only
J, nigra vs. J, regia .88 .71
J, nigra vs. hybrids .91 .35

J, regia vs. hybrids .85 .57

 

53
black walnut values. Clearly, it will be necessary to sample more trees,
especially pedigreed Fl's and backcrosses, before either scoring system
can be used with complete confidence. Nevertheless, the generally high
correlations between the two systems should enable us to rely for the
most part on the morphological hybrid index; the more expensive chromato—
graphic techniques and calculations of diagnostic values will probably

be used only when necessary to resolve doubtful classification.

CHAPTER IV

PLANTATION PERFORMANCE

 

Seedlings from 17 of the putative hybrid walnuts have been out-
planted in small replicated progeny tests in Barry County, Michigan and
Alexander County, Illinois. Twelve seedlots are common to both planta—
tions, but each lacks standard or check seedlings, such as good black
walnut of local origin, against which to measure possible superiority
of the hybrid trees.

Height data from measurements taken in January, 1971, in Michigan
and July, 1971, in Illinois are presented in Table 13. The nominal
butternut x Japanese walnut F3.hybrids (NC-5835 and NC-5836) were among
the taller progenies in both states, while Persian x black walnut
hybrids were relatively short. Spearman's rank correlation test
(Snedecor, 1957) for height of the 12 seedlots common to both locations
was highly significant with rS = 0.83.

In an attempt to assess tree form, the trees in Michigan were
measured for leader deflection from vertical (indicating crook plus
lean); in Illinois we tallied the number of main stems, defined as being
at least 85 percent as tall as, and 2/3 the diameter of the leader.
According to analysis of variance, differences due to seedlot were
statistically significant in the Illinois plantation, but after the
Michigan data were adjusted to show leader deflection as a percent of

54

55
Table 13.--Relative height and tree form traits (family mean
as a percentage of plantation mean) of seedlings from

open-pollinated putative hybrid walnut trees

 

: Plantation location
NC- : Presumed Juglans taxon of : Michigani/ : Illinoisl/i
number : open-pollinated mother tree : HeightJ: Form : Height : Form

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5801 nigra x cinereag/ 116 75
5822 regia x nigra 63 81 67 72
5826 sieboldiana x cinereaé/ 100 86 95 95
5827 sieboldiana x cinerea 126 110 100 90
5828 sieboldiana x cinerea 93 114 96 127
5829 sieboldiana x cinerea 100 117 94 119
5830 sieboldiana x cinerea 91 149
5831 sieboldiana x cinerea 97 101 112 108
5832 sieboldiana 141 104
5833 regia x nigra 94 79
5834 cinerea 130 85 114 116
5835 sieboldiana x cinerea 110 98 136 117
5836 sieboldiana x cinerea 132 . 98 121 93
5842 regia x nigra 85 97
5843 regia x nigra 65 98 67 76
5845 nigra x sieboldiana 104 106 120 78
5846 regia x nigra 68 90 70 106
Mean height, cm. 111 220
Mean leader deflection (cm. per

cm. height) 0.125
Mean number of 'main stems' 2.32

 

J] Trees in Michigan plantations were 5 growing seasons old
from seed when measured; trees in Illinois were 6 years old.

2] Probably J, nigra, see page 32.

J] Probably J. cinerea, see page 30.

56
total height, there was no appreciable difference among seedlots
(Table 13). Ranking among the families for the two form characters
was not closely correlated (rS = 0.33).

Seedlot NC-5822, comprised of seedlings from three Persian x black
walnut trees in Pennsylvania, had the least leader deflection in
Michigan and fewest main stems in Illinois. But since these seedlings
were also the shortest in both plantations, they don't seem to offer
much promise as timber trees. The correlation between height and form
was quite weak, with r = +.l9 in Illinois and +.27 in Michigan, so there
should be a respectable opportunity to select and breed for improvement
in both traits simultaneously. For instance, NC-5836, a butternut x
Japanese walnut F3(?) family, grew notably taller than average in both
plantations with somewhat better form (less than average leader deflec-
tion and fewer than average main stems). Other families also performed
well, including NC-5801 and NC-5845 in Illinois. As previously men-
tioned, the parent tree for NC—5801 is apparently a black walnut rather
than a hybrid; progeny of this tree also grew well in earlier tests in

Michigan.

CHAPTER V

CONCLUSIONS AND SUGGESTIONS FOR FURTHER RESEARCH

 

The research reported in this paper has involved a certain amount
of 'debunking', since some of the hybrid walnut trees and nuts pointed
out or sent to me have been found to be pure Juglans species. My
results tend to verify the generally accepted conclusion that naturally
occurring walnut hybrids are quite rare. Continued skepticism regarding
putative hybrids appears to be justified, especially when hybridity is
postulated on the basis of nut form that is unusual or perhaps inter-
mediate between presumed parent species. Fruit shape varies greatly
within Juglans species and many taxonomic forms have been described and
named on the basis Of little more than distinctive shell thickness or
ridging (Rehder, 1940). Other less variable traits of vegetative mor-
phology need to be incorporated into any satisfactory system of hybrid
analysis.

On the other hand, many of the advanced generation seedlings that
I studied still cannot be identified with certainty. As they mature,
flower and fruit characteristics can be added to hybrid indexes and more
precise determinations should be possible. Since I had to rely on vege-
tative traits to characterize the immature trees of the presumed hybrids,

it was necessary to use the same traits when constructing hybrid indexes

57

58

based on pure species. But with the exception of the twig key (Harlow,
1948) used to distinguish butternut from black walnut, none of the
references that I have seen (Funk, 1966) attempts to differentiate
Juglans species without reference to flowers and fruit. Future efforts
to identify putative hybrids should include the use of multiple samples
of leaves within trees and trees within families since hybrid seedlots
have been found to be quite variable in both morphological and bio-
Chemical analyses.

The increased variability of hybrid populations is basically desir—
able since it can reasonably be assumed to result from increased genetic
diversity. And increased diversity is the logical objective of hybridi—
zation programs. The examples of hybrid progeny with greater than aver—
age height growth and better than average form are encouraging since
rapid growth and poor form have been shown to be strongly correlated in

Juglans nigra progeny tests (Bey, g£_§J,, In press). If recombinations

 

which yield simultaneous improvement in both traits prove to be common
among hybrid walnut progeny, an expanded program of hybrid breeding will

be easily justified.

LITERATURE CITED

 

JAN

 

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Alston, R. E. and Simmons, Janiece.
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Alston, R. E., Turner, B. L., Lester, R. N., and Horne, David.
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60

Carter, Lynne C. and Brehm, Bert G.
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Clifford, H. T.
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15-21.

Whitson, John, John, Robert, and Williams, Henry Smith, eds.
1915. The production of a quick-growing walnut. In: Luther
Burbank—-His Methods and Discoveries and Their Practical
Application 11:193-237, illus. New York and London: Luther
Burbank Press.

Williams, A. H.
1955. Phenolic substances of pear-apple hybrids. Nature 175(4448):
213.

Wright, Jonathan W.
1962. Genetics of forest tree improvement. FAO Forest. and Forest
Prod. Ser. 16, 399 pp., illus. Rome.

Yablokov, A. S.
1936. [Selection of walnuts for rapid growth and winter hardiness.]
J. Moscow Sci. Res. Inst. Forest. 2:7-42. [In Russian]

 

 

VITA

David Truman Funk

Candidate for the Degree of
Doctor of Philosophy
Final Examination: October 14, 1971

Guidance Committee: Drs. M. Wayne Adams, August DeHertogh, James W.
Hanover, Stanley K. Ries, and Jonathan W. Wright, Chairman.

Thesis: Identification and Performance of Juglans Hybrids

David T. Funk was born in Greensburg, Indiana on October 17, 1929,
and spent his youth in small southern Indiana towns where his father
was a school teacher and administrator. He was educated in the public
schools, graduating from the high school at Osgood, Indiana. Funk
received B.S. and M.S. degrees in Forestry from Purdue University in
1951 and 1956. Mr. Funk married the former Nancy Lou Ross in 1956; they
have a daughter, Sara Jane, age 8.

Funk has worked continuously for the U.S. Forest Service for the
past 20 years except for active duty in the U.S. Navy from 1951—1955 and
time spent at graduate school. He has been assigned to silviculture and
tree improvement research projects in Ohio, Indiana, and Illinois and has
authored more than 25 publications on reforestation, propagation, and
genetics. He presently serves as Sponsoring Scientist for two Public Law
480 tree breeding research.projects in Yugoslavia.

Funk is listed in the 1969 edition of "American Men of Science."

He is a member of the American Association for the Advancement of

65

66

Science, the American Society of Plant Taxonomists, the International
Plant Propagators' Society, the Society of American Foresters, Sigma Xi,
and Xi Sigma Pi. He is presently chairman of the Society of American
Foresters national tree improvement committee, the Central States Tree
Improvement Conference, and the tree improvement committee of the walnut

Council.

 

APPENDIX

 

Table A—l.—-Morphological

67

characteristics of all Juglans species

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Flowers‘ ‘ fl Fruit 41 Nut
: : a; f
A 0 . o m o
e . . II o ,
Q .0 b0 0
V . .‘D .51"! .
. Q) .> p u-H .
L . u . /\ m o u ,
u ° CU -r\m u 0 .
OD .r—i .0V (0 A E Q. .
q .r—q .v :1 A 8 0') H o A
G) ,H . U H E (J VCU . E
.--q .4.» MA :3 U U v ..C - U
o (DU) .53 0) \ V Q) a) . V
a '-H u -o c) m ,c U .
~H . 9.0 .H m #J x: u m . . .c
:3 2% ~33 "a a ‘2: “a 8’8“ 3.:
Juglans m : 0.4 n4 3 t. H m g m : .H
species 0 , z m :0 0. LI: 3 .4 V m '0": 3 ‘
nigra 5-12 2— 5 l l- 3 3.5-8. 4 5-6. 5 2.8-5.
microcarpa 7 11 l— 3 1 l.2-2.2 2 l -l.9
9, var. l.8—2.5
Stewartii
major 7-17 1- 4 2 1.9-3.5 1.8-3.
m, var. 1- 3 l 3.6 4 3 3 -4.0
glabrata
hindsii 8—10 1- 3 2 3. —5. 1
californica 5— 7 l- 3 2 l. —3.2 3 3-3.6 2
hirsuta 6—10 1- 3 3 3. —4 7 4-5 2.5—4
pyriformis 18-22 4.3-4 7 4.5-5. 3-4 3.5-4.5
mollis 8-16 1— 3 l 3 -5 3.5-6 2 2.5—4.5
olanchana 15-30 2- 5 O 4.3-5.5 4.7-5.8 3.4—4.5
9, var. 3.:: 2 2.4-3.5
Standleyi
steyermarkii 3 3 4 4.5 5
neotropica 10-25 5-10 1 3 5—6. 3 5-6 3 3. -4.5
venezuelensis 5—10 6- 7 3 4 2.3
boliviana 10—16 3— 6 3-4 3.5-4.5
soratensis 15 5 4 5 5 5 3.1—3.4
jamaicensis lO—l3 5— 8 1 2 6-4. 3.5 3 1.9—3.1
australis 6-15 3- 6 1—2 2 5-3.5 3 -3.3 l 1.7-3.0
regia 5—15 1— 5 'O l— 3 4 —5. -4.4 O 2.3-3.9
cathayensis 20-30 3 6—10 3 -4.5 5
mandshurica 10 5—10 4.5-5.5 5
stenocarpa 5
sieboldiana 10-30 10-20 3 5412 2 5-3.5 5
g, var. 2— 6 3.5-4.5 4.5—5.0 O 3
cordiformis
cinerea 5- 8 5— 8 3 2— 5 4 —6.5 5

 

68

Table A-1 (Cont'd.)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

: Leaves : Branchlets : Mature
: : :r~ : : >. : : tree
,4 m
: : :v4 : : ur~z : :
u . ucn 003 o
: ; m u ; :CL :os~:~/o :,J : : -
. .4 m »\ . ch —4 >»
:m :tH.o : E : >~2CLU:$4P4: c : rx : mr~
> m a o 0 sun 0 o co m u'o
:rx' 0:3: v Leaflet :x: :r-4>»:u 3&4 zoom
.33*Jc.1: . .V.$®8 .V .3 .V3
.\/ ' ' u ’E’ ,\ ' o ° 0' .' La’M o ' u u
2 U: z : 01: U E 2Q:MH:U§:O§: E :Ou
m c c xx 0 to o m 0 r4 0 \x .4 m
:5 o:E§:§ : o \z :.c :594:~4Lt: o u: : o
0:) a .4 .c m ()0 1:5: o.o .4 u .
:H m:E§:E : u .c : L: :0 : : .c : g
.001-H +1 “-4 on u a; on: C1 a $3.3 00 .3
1251335 :m.o:c::§ : m c -o : m :wibm own: u u: ~4 : u o
-a a *4 o o -H ca ~13 u w wdCU o m a
species :omz:z: ..1 ..J :2 :cozs-«omauzm-o: :1: :mn
nigra 3 13 23 30-70 6-17 3 -6.2 3-4 0 g-B Tan 50 B
microcarpa 1 13 31 10-23 4- 8 1. —1.5 4 O r-Y Lt.B 10
E: var. 17 32 1.1—1.9
Stewartii
.major 2-3 9 19 30-36 3— 9 1.7—3.4 4-5 0 r-B Tan 20 gB
.2: var. O 9 17 30-45 8—15 2.3—5.5 5 O
LlabELEE
hindsii 1 15 23 6-10 0 15-25
californica O 11 19 3O 4- 7 1.5—2.2 2 O B Lt.B 16
hirsuta 3 14 23 15 2.5-6 3-4 Lt.B large
pyriformis 1 18 31 40-60 10-15 3 -4.5 B Lt.B tall
mollis 2 8 16 6-15 3 -5 4 dk.B Lt.B med.
olanchana 1 17 23 45-65 14—17 5 -6 5 0 Tan 55 B
‘9. var. 3 19 12 4 -4 5 4-5 0
Standleyi
steyermarkii 3 16 18 60 14—17 5 —8 4 17 G
neotrgpica 3 12 19 34—60 10-16 4 —8 2-3 Lt.B 30 B,G
venezuelensis 2 16 20 43 . 9-11 3.5-4 5 low
boliviana 1 14 18 13-17 5 -6.5 6 O Lt.B 35 dk.B
soratensis 1 15 17 42-44 8-11 3.5-5.5 3 Lt.B 9-10 1t.G
jamaicensis l 12 22 30-55 7-11 2 5—4.5 2-3 Lt.B 45
australis 1 14 20 30-64 8-16 3 —5 3 0 r—B Lt.B 7‘18
regia O 5 13 20-45 8-17 4.0-7.3 3-5 0 b-G Tan 30 G
cathayensis 3 9 17 40—90 8-22 5. - 1—2 + 12—25 C
mandshurica 3 9 17 30-40+ 7—18 4 - 2-3 + Brn. 20
stenocarpa 1 9 17 +
sieboldiana 2 9 21 55-60 8-18 3 -7 3-4 + Tan 20
.§° var. 2 9 17 35-45 11-15 4 -6 2-3 +
cordiformis

 

cinerea 3 11 19 6-12 3 -5 3-5 + dk.B 30 S

 

 

 

69

Table A—la.-—dgglans seed trees and clones used as sources

of experimental walnut seedlings and grafted trees

 

 

 

Number Plant material and origin

846 g, cinerea herbarium specimens from planta-
tion MSFGP 4—62, Fred Russ Forest, Decatur,
Mich.

1101 i, nigra seed, Stone Co., Ark.

1102 .1. nigra seed, Scott Co., Ark.

1601 d, nigra seed, Union Co., 111.

1605 g, nigra seed, Will Co., 111.

1708 g, nigra seed, Perry Co., Ind.

1709 g. nigra seed, Greene Co., 1nd.

1803 g, nigra seed, Delaware Co., Ia.

1805 g, nigra seed, Polk Co., Ia.

2004 g. nigra seed, Laurel Co., Ky.

2005 {. nigra seed, Madison Co., Ky.

2601 .3. nigra seed, Scott—Smith Co., Miss.

2602 .1 nigra seed, Chickasaw Co., Miss.

2708 g. nigra Seed, Bates Co., Mo.

3101 1, nigra seed, Caldwell Co., N. C.

3102 g, nigra seed, Graham Co., N. C.

3105 if nigra seed, Buncombe Co., N. C.

3803 _i. nigra seed, Union Co., Tenn.

3806 .i° nigra seed, Hardin Co.. Tenn.

3901 g, nigra seed, Freestone Co., Tex.

4101 1. nigra seed, Rockbridge—Amhurst Co., Va.

5801 i. nigra (?) seed, Cass Co., Mich.
=MSFG 848)

5802 g, nigra x cinerea o.p. seed, Cass Co.,
Mich.

5810 .1. regia seed, Hardy Russian strain P.I.
264373

5811 _i. regia seed, Hardy Russian strain P.I.
264376

5816 i. regia seed from 6 sib trees originating
in Stryj, Galicia

5822 _i. regia x nigra o.p. seed from 3 trees
near Lancaster, Pa.

5826 g, cinerea (2) seed, Berrien Co., Mich.
(~MSFC 841)

5827 is sieboldiana x cinerea o.p. seed, Berrien
Co., Mich. (=MSFG 862)

5828 J. sieboldiana x cinerea o.p. seed, Berrien

 

65., Mich. (ansrc 863)

 

70

Table A-13 (Cont'd.)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Number Plant material and origin

5829 g, sieboldiana x cinerea o.p. seed, Berrien
Co., Mich. (=MSFG 864)

5830 l. sieboldiana x cinerea o.p. seed, Berrien
Co., Mich. (=MSFG 865)

5831 g, sieboldiana x cinerea o.p. seed, Berrien
Co., Mich. (=MSFG 866)

5832 g, sieboldiana seed, Berrien Co., Mich.
(=M5FG 867)

5833 .13 regia 'Colby' x nigra o.p. seed of hybrid
walnut cv. 'Illinois xx'. Parent probably
pollinated by g. regia.

5834 1. cinerea seed from pooled progeny of MSFG
842, Kalamazoo Co., Mich.

5835 1. sieboldiana cordiformis o.p. seed from
pooled progeny of MSFG 852, Kalamazoo Co.,
Mich.

5836 i. sieboldiana cordiformis o.p. seed from
pooled progeny of MSFG 858, Kalamazoo Co.,
Mich.

5837 g. sieboldiana cordiformis o.p. seed from
tree C-4O of seedlot MSFG 858, Kalamazoo
Co., Mich.

5839 g, regia x nigra o.p. seed, Berrien Co.,
Mich.

5842 g. regia 'Hansen' x nigra o.p. seed, Marion,
Ind.

5843 l. regia 'Pomeroy' x nigra o.p. seed, Marion,
1nd.

5844 i. regia (Carpathian) x nigra o.p. seed,
Marion, Ind.

5845 1. nigra x sieboldiana cordiformis hybrid
cv. 'Leslie Burt' o.p. seed

5846 g, regia x nigra o.p. seed, Genoa, Ohio

5848 g, nigra x sieboldiana cordiformis o.p. seed
from a second-generation hybrid, Genoa, Ohio

5850 i. regia 'Alpine' o.p. seed

5851 i. regia 'Crath 5' o.p. seed

5852 {. regia 'Jacobs' o.p. seed

5853 g, regia 'McKinster' o.p. seed

 

71

Table A-la (Cont'd.)

 

Number

5854
5858
5859
5862
5880

5881
5892

5899

5900
5902

5903
5904
5905
5906
5907
5908
5909
5919
5920

5921

5922
5924
5925
5926

5927

Plant material and origin

.i° regia o.p. seed from a 'Crath 5' seedling
l. cinerea commercial seed of unknown origin
J. sieboldiana commercial seed of unknown

origin

g, nigra x regia
1nd.

grafting scions, Linton,

g: nigra x regia o.p. seed, Coloma, Mich.

. nigra x regia

b o

 

HuHu£findtflflqKa

‘dfldflq

France

o.p. seed, Coloma, Mich.

regia seed, Shogram, West Pakistan,
°30' N, alt. 8500 feet
. regia seed, Lahore, West Pakistan,
4°35' N, alt. 6000 feet
sieboldiana seed, Tokyo, Japan
. regia seed, Sarlat-Dordogne, France

regia seed, Brive(CorrEze), France
. regia seed, Vézére Valley, France
regia seed, Terrasson (Dordogne),

g. regia seed, Saint-Rabiner (Dordogne),

g, regia seed, Ayen (Corréze), France

g, regia seed, Terrasson (Dordogne), France
g, regia seed, Payzac (Dordogne), France
J

._. regia x nigra
grafting scions

ing scions

g, regia x nigra
grafting scions

g. regia x nigra
ing scions

g, regia x nigra
41-24-8)

g, regia x nigra
41—24—9)

i. regia x nigra
41-24-12)

g, regia x nigra
38-96-3)

hybrid cv. '3rd Avenue'
hybrid cv. 'Galley' graft-

hybrid cv. 'Stegalli

hybrid cv. 'Haig' graft-

grafting scions (-Beltsville
grafting scions (-Be1tsville
grafting scions (=Be1tsville

grafting scions (eBeltsville

72

Table A-la (Cont'd.)

 

 

Number : Plant material and origin

5928 J. regia x nigra (o.p.) grafting scions
(IBeltsville A2)

5932 J. regia x nigra (o. p. ) grafting scions
(-Be1tsville 49- O3— -11)

5933 J. regia x nigra (o. p. ) grafting scions
{-Be1tsville 49- O4— -3)

5934 J. regia x nigra (o. p. ) grafting scions
(‘Beltsville 52-06- -1)

5935 J. regia x nigra (o. p. ) grafting scions
(-Be1tsville 49-10-5)

5936 regia x nigra (o. p. ) grafting scions
(=Be1tsville 49-10-11)

5937 . regia x nigra (o. p. ) grafting scions
(-Beltsville 50-11-5)

5938 J_. regia x nigra (o. p. ) grafting scions
(-Be1tsville 50-12——5)

5939 J. regia x nigra (o. p. ) grafting scions
(-Be1tsville 51-01- -4)

5940 J. regia x nigra (o. p. ) grafting scions

(-Be1tsville 51- ~02-3)

5941 J_. regia x nigra (op .) grafting scions

(- -Be1tsville 51- 02- -4)

J, regia x nigra (o.p.) grafting scions

(-Be1tsville 52-06- -4)

5943 regia x nigra (o. p. ) grafting scions
(=Be1tsville 52-10- -7)

5944 regia x nigra (o. p. ) grafting scions
(=Be1tsville 55-81- 5)

5945 .J. regia x nigra hybrid cv. 'Coye' o.p.
grafted nursery stock

5942

5948 .J. regia x nigra hybrid cv. 'Norris'
o.p. grafted nursery stock

5949 (J, regia x nigra F2) x 'Norris" grafted
nursery stock

5951 J, regia x nigra hybrid cv. 'LaPlata'
o.p. grafted nursery stock (IBeltsville
42-14P-4)

5954 J, regia x nigra (2) hybrid cv. 'Hillgate'
grafting scions

5956 J, regia x nigra hybrid cv. 'O'Connor'
o.p. grafting scions (-Beltsville 49-05-5)

 

73

Table A-la (Cont'd.)

 

 

Number : Plant material and ongin

5957 J, regia x nigra hybrid cv. 'O'Connor' o.p.
grafting scions (-Beltsville 49-05-12)

5959 J, regia x nigra (?) hybrid cv. 'Fort
Hunter' grafting scions

6133 ‘J. nigra seed, Sewanee, Tenn.

6135 .J. nigra seed, Connersville, Ind.

6137 J, nigra seed, Abbington, Ind.

6138 J, nigra seed, Amana, Ia.

6143 J, nigra seed, Bedford, Ind.

6144 J, nigra seed, Ewing, Ind.

6145 J, nigra seed, Jasper, Ark.

6146 J, nigra seed, Franklin, Ind.

6148 J, nigra seed, Covington, Ind.

6149 J, nigra seed, New Carlisle, Ind.

6150 “J. nigra seed, Decatur, Mich.

6154 J, nigra seed, Orange Co., Ind.

6155 J, nigra seed, Crawford Co., Ind.

6156 J, nigra seed, Mendota, Ill.

6157 ‘J. nigra seed, LeRoy, Ill.

6159 ‘J. nigra seed, Montpelier, Ohio

6160 J, nigra seed, Crawfordsville, Ind.

6163 J, nigra seed, Linden, Ind.

6166 J, nigra seed, Elizabeth, Ind.

6170 J. nigra seed, Wooster, Ohio

6171 J. nigra seed, Troy, Ohio

6173 J. nigra seed, Columbus, Ohio

6174 ‘J. nigra seed, Delphos, Ohio

6175 J, nigra seed, Fairfield Co., Ohio

6179 J, nigra seed, Paris, Ky.

6182 J, nigra seed, Saline Co., Mo.

6183 J, nigra seed, Palmyra, Mo.

6190 J, regia cv. 'Illinois 3' grafted nursery
stock

6191 J. regia cv. 'Lake' grafted nursery stock

6192 J, regia cv. 'Helme 2' grafted nursery stock

6193 J, regia cv. 'Caesar' grafted nursery stock

6194' J_ regia cv. 'Colby' grafted nursery stock

6195 J, regia cv. 'Hansen' grafted nursery stock

 

74

Table A-la (Cont'd.)

 

 

 

 

 

Number Plant material and origin

6196 J, regia seedlings, unknown origin

6197 s 6190

6198 J, sieboldiana cordiformis cv. 'Rhodes'
grafted nursery stock

6199 J, sieboldiana cordiformis cv. 'Wright'
grafted nursery stock

6200 J, regia x nigra o.p. seed

6201 J, nigra seed, Otoe Co., Nebr.

6202 J, nigra seed, Fairfield Co., Ohio

6208 J, regia x nigra hybrid cv. 'Lorenz"
scionwood

6210 J. regia seed, Barkob Research Range,
India. Altitude 7000-8000 feet

6471 J. cinerea herbarium specimens, Kellogg

Forest plantations, Augusta, Mich.

 

 

Table A—2.——Juglans nigra morphological data

75

 

 

 

 

 

 

 

(l) (2) (3) (4) (5) (6) (7) (8) (9) = (10)
J LeaflegfiJ :
U) i
,1.“ a Blade 8,\ 3 :
<0 G) O . 7‘ °H E 3 .
.3 °‘ T3 '5 ' .s ‘8 ° 3 I
. (0‘44 H 00/\ 4—1 A $4 1.4 E A-

Seed source : Sample 0 $3 ‘8 S E 3:", E 23 8, a a:

No. :Location: size1 2 "”3 ‘3‘ "7 V +3 VJ m V U V: (5)/(4)
1601 111. 4,6 18.0 2.8 136.8 50.8 4.5 5.3 0.16
1605 111. 2,6 16.5 4.0 102.5 39 5.5 3.5 .24
1709 Ind. 2,6 17.0 3.0 142.5 54.5 3.5 3.5 .18
1805 la. 2,6 15.0 2.0 100.5 39.5 5.0 6.0 .13
2004 Ky. 2,6 15.0 2.0 135.5 53.5 5.5 8.5 .13
2005 Ky. 2,6 17.5 5.5 135.0 54.0 5.0 6.0 .32
2602 Miss. 4,6 18.5 3.5 121.5 48.0 5.3 3.8 .19
2708 Mo. 2,6 15.5 3.5 112.5 43.0 5. 5. .23
3101 N.C. 2,6 16.0 3.5 117.5 40.5 4.5 3.5 .22
3102 N.C. 2,6 17.0 3.0 160.0 56.5 4. 5. .18
3105 N.C. 3,6 17.7 3.7 115. 43.0 6. 5.0 .21
3803 Tenn. 3,6 16.0 3.3 121.3 51. 4.3 3. .21
3901 Tex. 3,6 20.3 5.0 113.3 41.3 4.3 6.3 .25
1101 Ark. 1,6 23.0 6.0 117. 47. 7. 4. .26
1803 Ia. 2,6 20.5 4.5 106. 38. 4.5 5. .22
2601 Miss. 1,6 23.0 5.0 105. 39. 5. 0 .22
3806 Tenn. 1,6 23.0 4.0 152. 58. 5. 5. .17
4101 Va. 2,6 22.0 6.0 124. 46.5 5. 9. .27
6138 Ia. 6,10 18.2 5.3 104.2 35.3 4.3 5.7 .25
6156 111. 0,9
6157 111. 6,5 18.5 4.5 112.5 38.3 3.7 4.7 .24
6159 Ohio 6,8 19.3 4.3 91.5 33.2 6.5 3.3 .22
6160 Ind. 6,9 19.3 4.0 104.3 37.2 4.5 3.3 .21
6163 Ind. 6,9 17.8 4.3 104.7 36.5 4.8 4.7» .24
6166 Ind. 6,5 16.0 4.7 103.3 37.3 4.3 3.2 .29
r 2! 18.36 4.1 118.3 44.2 4.88 4.7 .218
32 6.27 302.3 .67 .002
s E] .51 3.55 .17 .009
05 .13 .15 .17 .21

 

J/ First value gives N for columns 4-14; second value for columns
15-19.
_2_/ Unweighted mean for columns 1—16; weighted for columns 17—19.
_3_/ Standard error of seedlot means.

 

76

Table A—2 (Cont'd.)

 

 

 

(1) (2) (11) :"(12) (13):“,(14) (15) : (16): (17): (l8):(19)
° :0 E; ' : : :
x .suu3
0 ':m w
'o .anm m n
6 ° tom 6 o
#4 .lrdU a .4
'w :6 .U o
H .O'U‘H G) U H U)
U 'CCH H (U Q
.4 .QHUD CD 0 B
‘H '0 CD (I) U) 00 0) O
Q 0'0)“ -.-4 H m . .1:
° 0 ' 'GJGJ‘HS ..C ‘H ' ' LH U
Seed source : '4 : :Base :vgggg g '3 . :Pith : 3";
No.:Location:(6)/(Z):(9)[(6):shape:9”**‘4 3‘ ‘“ :Scurf:color:~4 “
1601 Ill. 2.69 0.039 3.3 9.8 1.67 1.33 0
1605 Ill. 2.63 .034 4.0 9.0- 2.00 .90 0
1709 Ind. 2.61 .025 4.0 8.5 1.92 1.83 0
1805 Ia. 2.54 .060 4.5 9.0 2.08 1.50 0
2004 Ky. 2.53 .063 3.0 8.5 1.83 2.30 0
2005 Ky. 2.50 .044 3.0 9.0 2.17 1.70 O
2602 Miss. 2.53 .031 3.8 9.3 2.08 1.83 0
2708 Mo 2.62 .044 4.5 11.0 2.08 0.62 0.08
3101 N.C. 2.90 .029 4. 8.5 1.92 1.42 0
3102 N C 2.83 .031 4. 11.5 1.58 1.90 0
3105 N.C. 2.67' .043 3.0 10.3 1.92 1.80 0
3803 Tenn. 2.38 .025 3.3 10.3 2.00 1.40 0
3901 Tex. 2.74 .056 2.3 14.0 2.33 1.33 .17
1101 Ark. 2.49 .034 3. 9. 2.08 1.17 0
1803 Ia. 2.79 .047 3.5 11. 1.92 1.70 .08
2601 Miss. 2.69 0 4. 13. 1.75 2.08 O
3806 Tenn. 2.62 .033 5. 11. 1.83 1.58 .08
4101 Va. 2.67 .072 4. 10.5 1.92 2.17 0
1102 Ark. 2.33 1.42 0
1708 Ind. 2.00 2.00 0
6133 Tenn. 1.22 3.00 1.83 1.14 .33
6135 Ind. 2.14 2.43 1.40 0.92 0
6138 Ia. 2.98 .055 3.8 10.0 1.20 3.05 1.60 1.43 0
6156 Ill. 1.22 2.87 1.83 1.33 .11
6157 Ill. 2.94 .042 4.0 10.5 1.60 2.80 1.90 0
6159 Ohio 2.76 .036 4.2 9.8 1.17 2.83 1.50 1.50 0
6160 Ind. 2.80 .032 3.2 12.0 1.7 2.5 1.4 .70 .05
6163 Ind. 2.87 .045 4.5 9.2 1.4 2.8 1.4 .75 .17
6166 Ind. 2.77 .031 3.2 12.2 1.60 2.90 1.90 0
6176 Va. .70 2 60 1.80 .25 .05

 

 

Table A-2 (Cont'd.)

77

 

 

 

 

(1) (2) (11) :"(12) (13):h(14) (15) : (16): (17): (18):(19)
: : O '2 ' : : z
X :EHCU
Q) :30) U)
“U . (03!!) U) H
a ' I 00) OJ 0
...4 - lr—IU f: H
'0) CO '0 O
u . U'UN—J (D U M U)
0 ‘ aczu H m m
H :OJCUS Q) U a
M 0 mm a: on 0') O
‘0 , -c0H 'H H m . ,c:
' 0 ‘main-(r: ,2 '1-4 ’ ‘4.) 0
Seed source : r4 . :Base "3333 g '3 :Pith : 8 g
No.:Location:(6)/(7):(9)/(6):shape:9*3’1“ § J ff :Scurf:color:~4 U
MSFG
178 Mich.? 2.14 1.14 .05
6173 Ohio .5 2.5 1.0 1.0 0.5
6179 Ky. 3.0 3.0 1.8 0
6143 Ind. 3.0 3.5 1.8 2.0 0
6202 Ohio 2.5 2.5 2.5 1. 0
6145 Ark. 4. 2.5 1. 0
6144 1nd. 2.5 2.5 2.0 1.5 0
6170 Ohio 2.0 2.8 2.0 1. 0
6146 Ind. 1.5 2.5 1. 1.0 0
6148 Ind. 0.5 2.5 1.8 2. 0
6174 Ohio 3.0 2.5 1.8 0.8 0
6154 Ind. 1.5 2.8 1.5 3. 0.3
6183 Mo. 3.0 3.0 2. 2. 0
6155 Ind. 3.0 2.8 1.3 1.5 0
6201 Neb. 2.0 2.5 2.0 1.3 0
6149 Ind. 3.5 3.5 1.5 1.3 0
6171 Ohio 3.0 3.0 1.3 1.3 0
6150 Mich. 2.0 2.5 1.8 2 0 0
6182 Mo. 2. 2.5 2. - 0
i Z/ 2.69 0.040 3.71 10.29 1.96 2.76 1.80 1.45 0.040
32 .024 .0002 .404 2.16 0.11 .272 .010
S52 2/ .031 .003 .130 .300 0.047 .079 .014
cv .06 .38 .17 .14 .18 .36 2.45

 

 

Table A-3.-Jgglans regia morphological data

 

:(14): (15): (16)

v1(13)

(12)

(11)

(9) : (10)

(8)

(7)
Leaflet

(6)

:(3) :(4) :(5)

(2)

(1)

O.

O.

 

semoqorzn
aeos 3231

1010:
“31d

.0 00 so 0. 00 '0

aouaosaqnd
adeqs
3863

(911(6)

(5970.12 (697(7)

0. 0. O. O. .0

aanasaang-

suorasazas
0"”!

so so 00 so to so

“JPIM

q38ueq

uorarsoa
O. O. Eg";’1:
/813I;831

azrs
atdmss

O. O. O. O. I. 0.

Seed source
Location
or cultivar

No.

 

 

4.3 0.2

025
.124
.029

2.25
2.26
2.05
2.41

.041
.111
.060
.111

3.0
16.

7.4 0.3 121.3 54.0 1.0

Soviet Union 3

5810

4.0 0.5

129. 57. 2.

8.3 0.5 95.8 46.7 0.9

1.

Soviet Union
Galicia

5811

1.8 0.3

4.0 0.5

2.8

6

5816

.053

54. 0

0 1:3().

'Alpine' open

5850

pollinated

78

3.4 0.3

:BIF() ()0-5
2.5 0.4

3.0 0.5
033

.023
.02

.065

1.97
2.53
2.02
1.80

.111
.170
. 44
.158

4.3
2.7
3.0

122. 62. 0
8.8 1.5 128.5 50.8 0.7

1.
9.5 1.5 114.5 63.5 1.3

9.0 1.3 116.7 57.7 1.0

1
6
3

'Crath' seedling 2

'Crath 5' o.p.
'Jacobs' o.p.
'McKinster' o.p.

5851
5852
5853
5854

3.8 0.4

.018
.042
.020
.042

2.11
2.18
2.21
1.99

.205
.144
.144
.111

2.2

7.8 1.6 122.8 58.2 1.9
9.0 1.3 129.5 59.5 1.0

5
4
3

Pakistan

W.

5892

3.8 0.3
2.7 0.3
3.3 1.0

5.5

Pakistan

France
France

W.

5899

9.0 1.3 100.3 45.3 0.7

5902
5903

9.0 1.0 107.8 54.2 0.3

6

3.3 0.5
3.7 0.7
3.6 0.4

4.0 0.3

045
.050

2.11
2.06
1.98
2.07

.111
.144
.133
.188

:5'.‘)
6.3

3

France

5904
5905
5906
5907

9.0 1.0 110.0 52.3 1.3

9.0 1.3 125.7 61.0 1.7

3
5

France
France

.050
.074

6.0

9.0 1.2 119.0 60.0 1.4

7.0

2 9.0 1.5 94.0 45.5 2.3

France

2.00 .078 3.0 0.5
1.98
2.06
2.29

.111

8.5

9.0 1.0 109.0 54.5 0

2

France
France

5908

1.3

3.0 0.5
3.7 0.9

.043
.034
.015

118

.149
.111

4.5
:30‘7

8.5 1.0 104.0 52.5 1.4
9.4 1.4 110.2 53.6 0.5

4
14

Commercial

5909
6196
6210

0

3.0

1. 133. 58. 1.5 2.

9.

India

 

.mnmoa uoavmom mo nonnm unannoum \M

.qdlsw ”555400 EOHN uaflhflwmfiv G023 0H fig MH mgdoo MON 2 60>.fiw flfifidxr UGOUQW \HHI

 

 

 

79

 

 

 

0m. 00. 0H. 00. 00. 0m. 00. OH. 00. va
00. 00. NH. 000. 00. 000. «a. m.m «H. \N m
00. 00. 00. H00. 00. N00. 00. «.mmn 00. N0
0 00.0 00.0 0.0 000. 0n.~ 00H. ms. N.0HH -.m m
o 0.0 0.0 0.0 000. NH.~ 00H. 0.0 0 0.00 0.0HH 0.H 0.0 0.0 .connom. 00H0
0 0.0 0.0 0.0 000. H0.~ 50a. 0.0 0 0.00 0.0HH 0.H 0.0 m.m .mnaou. 00H0
0 m.a 0.0 0.0 0H0. 0N.~ nan. 0.H 0 0.H0 0.~0 0.4 0.0 0.N .nmmooo. 00H0
0 0.0 0.0 0.0 H00. ~0.~ 000. 0.0 0 0.00 “.00H w.H 0.0 0.0 .N madam. NOH0
0 0.0 0.0 0.0 050. 0H.N NNN. 0.0 0 0.00 0.00H 0.0 0.0 0.m .oxma. HOH0
0 0.H ~.0 m.~ 000. 00.N 00H. ~.0 0 0.00 m.wHH 0.H N.“ 5.0 .m .HHH. 00.00H0
0 0.4 0.0 0.0 00H. 00.0 and. .MH 0 .00 .00a .4 0.0 0.H .Hoogom. 00H0
3 1" o .o. d u s so uA0¢\A0vu:v:0v"30\~0vu 3" s N” M u 1 dale.“ s S” nee/316 no " .oz
13” or." nnqe" u u Hanson..." enouaaulm” nonumuoq "
1.8 T.3 a. B s J 1 . D. u s e e 2
31.” on." ands. u n ”A"; "3" 8 "anand" monaomvoom
U. . 1 . S .3 . . . . B . B . u. . 3 . 3. I. IT.
03. . 3 . . . . 3 . 3 . . u. . r... 8./au
m o a n .1 o 3
a e u u u u H u u n .1 u o u u u u u s" "
81- o a. o O O O a O u 0 O O O /. O
. . 3. . . . . . 8 . . o . o o
. u u u u u " uoawsmq " n n
1650 1 Anne 1300. Anne” Anne 1 Anne 1 nose 1 Ass Ass Ass 160 Ame. Ase. Ame. 1N0 A40

 

A.p.u:oov 014 wapwfi

 

Table A-4.~~ggglans regia x nigra morphological data

 

 

 

(l): (3) :(4): (5) (6) (9) = (10) (ll) :(12):(13)
: : : : Leaflet : :

i 2 3 m. a

.- . . a . o . u

. . CD . G . 0' u . a

:0 :‘fi :~S : 3 I: 3 3 3 3

.v-i .H . u u I: . El. G . 0. O

'O.0°tH*H ..a no u - n - > . g a” o

.a a. a n . m a -c ,. n . 1. . . . . m d. .0

wow-vom-o Q, H .0”. :1 . .. . .mg. :3

No.:"‘ qr’: '4" :n‘: '4 3 :2 °° ' U :L4)/(3l:(5)/(6l:(8)/g5):‘n ": °‘
5822 8 9.9 1.0 118.4 49.8 2.1 6.5 .101 2.42 .055 4.3 0.4
5824 2 13.0 1.0 108.0 40.0 3.5 2.0 .077 2.70 .018 2.5 1.0

5839 1 7. 1. 85. 36. 2. 4. .142 2.36 .047 4. 0
5842 5 9.8 1.0 101.6 47.4 2.5 3.2 .102 2.15 .031 3.3 0.1
5843 6 9.7 1.0 107.8 53.2 1.3 3.3 .103 2.02 .031 2.8 0.1
5844 3 12.7 2.7 113.0 46.7 5 5.7 .212 2.42 .050 3.5 5.5
5846 5 10.6 1.4 116.0 49.2 2 9.2 .132 2.36 .079 3.9 0.4
5862 1 13. 2. 90. 41. 4. .154 2.19 .044 3. 0.5
5880 5 11.8 2.0 125.2 45.2 3.6 .169 2.77 .029 2.6 1.8
5881 4 11.5 2.3 96.0 42.3 4.0 .200 2.27 .042 3.5 6.4
5920 2 12.0 2.0 103.5 39.5 3.0 .167 2.62 .029 4.0 8
5921 2 12.5 2.0 123.5 45.0 1.5 .160 2.75 .093 3.5 5
5922 2 11.0 2.0 110.5 51.5 6.5 .182 2.15 .059 3.5 5
5924 3 11.7 2.3 112.0 56.0 6.7 .196 2.00 .060 3.5 5
5925 1 13. 2. 138. 49. 5. .154 2.82 .036 3.5 .0
5926 1 17. 5. 139. 53. 7. .294 2.62 .050 4. 0.5
5927 7 11.1 2.6 108.3 48.4 6.3 .234 2.24 .058 3.4 2.9
5928 3 12.3 2.3 153.3 68.0 5.3 .187 2.25 .035 4.5 2.0

5932 2 13.0 2.5 120.0 49.0 12.0 .192 2.45 .100 3.0 0
5933 4 9.8 2.0 96.8 44.0 5.5 .204 2.20 .057 3.4 0.1
5934 3 10.0 2.0 128.3 62.0 6.0 .200 2.07 .047 3.0 5.0
5935 5 11.0 2.0 98.8 37.8 7.6 .182 2.62 .077 3.4 5.8
5936 1 7. 0 109. 48. 14. .000 2.27 .128 4.5 0.5
5937 2 14.0 3.5 119.5 47.5 14.0 .250 2.52 .117 4.3 0.8
5938 2 10.0 2.5 107.0 41.0 10.0 .250 2.61 .093 4.5 7.8
5939 3 14.7 3.7 87.3 38.7 3.7 .252 2.26 .042 3.3 8.5
5940 3 17.7 5.7 86.0 33.7 4.0 .322 2.55 .046 3.7 7.7
5941 2 14.5 3.0 117.5 59.0 1.5 .207 1.99 .013 3.3 8.0
5942 5 20.0 6.0 97.8 31.8 4.2 .300 3.08 .043 4.1 7.1
5943 3 19.0 5.7 97.6 33.0 7.7 .300 2.96 .079 1.0 11.8
5944 1 15. 3. 113. 46. 5. .200 2.47 .044 3.5 6.5
5945 1 11. 1. 116. 58. 4. .090 2.00 .034 1.0 6.0
5948 2 10.0 1.0 106.5 51.0 3.5 .100 2.09 .033 2.5 1.0
5949 1 19. 3. 119. 44. 4. .158 2.70 .034 3.0 3.5
5951 2 11.0 2.0 83.0 35.0 4.5 .182 2.37 .054 3.5 1.0

 

8]

Table A-4 (Cont'd.)

 

 

 

 

(l):(2): (3) :(4): (5) (6) (7) (8) (9) (10) (ll) :(12): (13)
: : z : Leaflet : :
:\ 8: .. : : 3
cm (3 o. u o o C
:Q, :3 .3 _c I}: 3 : : 3
:10:C144 ' :3 :3 '5 :3: E : 0 §3 g
512:3 3 8 g; :3 : 5 S: a : : : :3.:: '3
_go “Wu-1H e >3 3 :2”:0 z(4)/(3):(5)/(6):(8)/(5):“”z 9‘
5954 3 12.3 2 3 116.0 48.7 3.2 2.0 .187 2.38 .017 4.7 1.2
5956 1 13. 2 97. 43. 2. 4. .154 2.26 .041 1.5 0.5
5957 1 9. 1 92. 34. o 3. .111 2.70 .033 2.5 0
5959 4 14.0 3 5 115 8 42.0 3.3 4.8 .250 2.75 .041 4.3 6.5
6200 1 15. 3 102. 43. 3. 5. .200 2.37 .049 2.5 0.5
6208 1 9. 1 95. 43. 1. 2. .111 2.21 .048 3.0 1.0
X 12.40 109.0 2.87 .180 2.41 .052 3.34 2.90
$2 9.12 233.0 2.01 .005 .08 .0007 .74 10.02
sxll .47 2.4 .22 .011 .04 .004 .14 .49
cv .24 .14 .49 .37 .11 .50 .26 1.09

 

.1/ Standard error of seedlot mean.

 

82

Table A-5.—-Weighted hybrid index (h.i.) values for seedling families

of Juglans regia, g. nigra, and g, regia x nigra hybrids

 

 

 

 

 

 

 

Juglans‘gégia : Jnglans regia
seedling gamilies 2* seedlingLfamilies
Accession : : Family : Accession : : Family
number : h.i. : mean h.i. : number : h.i. : mean h.i.
5810 123 5902 110
97 139
94 105 108 119
5811 126 126 5903 119
5816 95 82
119 108
78 109
120 134
111 102 109
92 103 5904 114
5850 118 118 113
5851 104 104 124 117
5852 110 5905 128
122 119
110 120 122
107 5906 114
132 124
127 118 107
5853 134 92
118 113 110
109 120 5907 119
5854 130 132 126
102 116 5908 109
5892 121 113 111
134 5909 108
138 105
113 113
112 124 124 113
5899 116 6210 121 121
107
119

133 119

 

83

Table A~5 (Cont'd.)

 

 

 

 

Juglans nigra : Juglans nigra
seedling families : seedling families
Accession : : Family : Accession : : Family
number : h.i. : mean h.i. : number : h.i. : mean h.i.
1101 273 273 4101 275
1601 231 272 274
232 6138 237
252 291
253 242 262
1605 253 260
241 ' 247 269
1709 212 281 267
238 225 6157 246
1803 254 249
279 267 252
1805 230 294
229 230 264
2004 223 274 260
224 224 6159 277
2005 257 266
245 251 242
2601 286 286 274
2602 230 262
225 288 268
253 6160 264
265 243 269
2708 243 266
‘ 256 250 248
3101 245 281
238 242 279 268
3102 253 6163 241
252 253 248
3105 245 243
273 262
258 259 252
3803 208 280 254
214 6166 259
266 229 271
3806 266 266 250
3901 278 263
287 276

291 285 270 265

 

 

 

84

Table A-5 (cont'd.)

.——.. - -

Hybrid seedling tamilies : Aflybrid seedling families

 

 

Accession : : Family : Accession : : Family
number : h.i. : mean h.i. : number : h.i. : mean h.i.
5822 127 5844 168
130 133 141
105 5846 120
123 126
141 124
149 130
144 140 128
129 131 5880 122
5824 180 128
141 161 127
5839 123 123 194
5842 126 225 159
132 5881 183
87 156
143 212
131 124 198 187
5843 108
83
122
102
92

142 108

 

85
Table A—6.—-Morphological data and weighted hybrid index for

Juglans sieboldiana and i. cinerea

”‘—

 

 

 

 

 

 

 

(1) <2) <3) (4) : <5) : <6) <7) <8) <9) <10) <11) (12)
: : ‘fLongest leaflet
\ i 7‘. .
H4) 0) ° ° to
..C U A . . (0
N u 0) E A ‘ (D ' ‘ 0) 0)
+4 60 c—i E . c: . . 0.. C
m c tn 0 \x a - o '0; - m '0
Seed source 61 ,9: 8 ,2 .4: V : If, E : ’5 z '23,, .3;
: Location : '3“ 4.. A "‘ :1 ‘5") f, : S 0 33A : q, ‘3
- 0r - S 85 o 8 8 3 =SS=BE=$ 8
No.: cultivar : 0 '4 V 2 ‘3“ 5" 3 :U’ “:53V : 9° '4
g. sieboldiana
5832 Michigan 6,1 15.7 3.5 146.2 55.7 3.1 3.5 2.8 2.8
5859 Commercial 9,9 57.2 18.0 4.0 154.1 58.2 4.6 6.2 3.9 3.9
5900 Tokyo 3,20 13.0 2.3 173.0 63.3 3.0 3.3 2.7
6198 'Rhodes' 6,16 40.2 12.7 2.8 141.3 57.5 3.3 7.0 2.1 3.5
6199 'Wright' 3,3 38.7 14.0 3.0 113.3 47.3 3.3 4.0 2.7 1.3
i 45.4 14.7 3.1 145.6 56.4 3.5 4.8 2.8 2.9
82 105.6 4.8 .43 471.3 33.8 .42 2.8 .43 1.3
s; 2/ 5 9 1.0 .29 9.7 2.6 .29 .75 .29 .57
cv 23 .15 .21 .15 .10 19 .35 23 40
g, cinerea
MSFG
846 Michigan 19,16 38.8 15.6 3.2 104.8 37.9 4.1 2.9 3.6 0.8
5858 Commercial 7,7 30.6 16.1 4.1 93.3 44.3 3 9 2.3 2.3 0.9
6471 Michigan 11,2 39.9 16.0 3.7 105.7 37.0 4 2 1.8 4.5 0.6
i 36.4 15.9 3.7 101.3 39.7 4.1 2 3 3.5 .77
82 2 25.8 .07 .20 47.8 15 8 02 30 1.2 .02
s; _/ 2.9 .15 .26 4.0 2 3 .09 .32 .64 09
cv .14 .02 .12 07 10 .04 .24 .32 .20

 

i av—ww

ll First value gives N for columns 4-15c, second value for columns
16-17.
_2_/ Standard error of seedlot means.

 

Table A—6 (Cont'd.)

86

 

 

 

 

 

 

(1) (2) (f3) (14) :(lSa) (15h) (15c)?(16):(17):(18)
><
Pubescence m
H "U
. ‘ O :HU):;:
/'\ A H (U0 'H
Seed source : b~ I\ Lea o I:C)E :
v v v U) U (DO 'U
: Location : \s ‘\ H ' M -H : .: z'H
or 2 9: a 6 8 5 =8u=i
No.: cultivar : ~1 ~/ D -9 ' m li-91-:'4“l :n
g, sieboldiana
5832 Michigan .024 .019 2.3 8.0 7.8 1. 5. 43.6
5859 Commercial .040 .025 0.9 6.4 5.4 1.1 2.7 42.9
5900 Tokyo .019 3.5 5.7 4.0 1.4
6198 'Rhodes' .050 .025 2.5 7.7 8.0 0.5 3.0 47.3
6199 'Wright' .029 .011 2.0 8.0 7.7 0.8 2.8 35.1
i .032 .020 2.2 7.2 6.6 ' 0.85 3.0
32 .0002 .00004 .88 1.1 3.2 .07 1.7
s-.£/ .006 .003 .42 .47 .80 .13 .58
Cfi .39 .33 .42 .15 .27 .31 .43
_ g3 cinerea
MSFG
846 Michigan .028 .008 2.3 5.9 3.7 3.7 3.6 18.6
5858 Commercial .025 .010 2.4 3.7 3.7 3.8 2.6 17.7
6471 Michigan .017 .006 2.4 5.5 3.0 1 3 4.3 21.4
i .023 .008 2.4 5.0 3.5 2.9 3.5
52 .00003 .000004 .003 1.4 .16 2.0 .73
Si 2] .003 .001 .03 0.7 .23 .82 .49
CV .24 .25 .02 23 .12 .48 24

 

 

Table A-7.--Juglans sieboldiana x cinerea morphological data

87

for traits selected fOr inclusion in hybrid index

 

NC

NC

NC

NC

NC

NC

NC

NC

NC

Accession number

5826
F2(?) seedlings

5827 - MSFG 862
F1 parent
F2(?) seedlings

5828 = MSFG 863
F parent
F2(?) seedlings

5829 - MSFG 864
F parent

F2(?) seedlings
F3(?) seedlings

5830 - MSFG 865
F1 parent
F2(?) seedlings

5831 - MSFG 866
F1 parent
F2(?) seedlings

5835
F3(?) seedlings

5836
13(7) seedlings

5837
F3(?) seedlings

:Sample:Lesflet:

Relative :

Rachis

:Pith

: size :1eng§h_:lgpsidednessigubescence:color: H.I.

5 88.4
3 133.7
10 96.0
4 124.5
7 112.0
4 120.3
6 98.0
5 91.6
4 102.8
4 112.0
4 130.8
7 112.9
8 108.5
6 106.2
9 103.3

.007

.013
.019

.031
.023

.007
.027
O 026

.010
.017

.006
.016

.018

.019

O 029

4.0

HUI bx:
Nuoo woo

uiuaox

NU!
UVUI

UU‘
O

4.0

5.0

4.5

4.0

hJP‘P‘
. 0
U1 UI

F‘P‘
O 0
~01»

F‘P*

1.9

2.5

2.0

15.7

 

 

88

Table A-8.--Position and description of spots on paper chromatograms

of leaf extracts of Juglans regia, J, nigra, and

 

 

 

 

putative g, regia x nigra hybridsl/
: J. regia : J. nigra : J. regia x nigra : 0016;377
: Zl: : : : : : UV (or visiblgl
Spot: -1— :RF-Z: -1 : RF-Z: R -1 : RF-Z : :
no.: RF : :RF : F : Initial NH3
1 .61 .00 .60 .00 .54 .00 via. yel
2 .73 .00 .66 .00 gold vis. yel.
3 .81 .00 dk. purp-brn vis. gra-brn
4 .33 .11 .35 .10 .36 .11 orange to via. orange
dk. brown
5 .46 .16 .52 .07 grown
6 .57 .24 .53 .20 .50 .23 purp-brn vis. tan
8 .45 .35 .46 .32 .49 .33 peach
9 .36 .47 .39 .39 .41 .46 brt. grn
10 .50 .42 .51 .40 .50 .44 lavender vis. lav-
gra
ll .61 .43 .59 .38 .57 .43 blu-gra
12 .74 .33 .72 .27 .65 .29
13 .44 .48 .46 .43 .48 .46 pink
14 .64 .47 .57 .45 via. tan
15 .72 .51 .72 .50 blue
16 .38 .52 .40 .50 .41 .54 grn-gra
17 .46 .56 .48 .49 .48 .55 via. gra-
grn
18 .66 .50 gray
19 .55 .62 .57 .58 .53 .59
20 .68 .64 .68 .64 .64 .67 blu-wh
21 .22 .61 .23 .61 blue
22 .25 .60 .29 .53 .25 .56
23 .37 .61 .37 .63 .36 .69 faint blue gray
24 .44 .64 .44 .63 .45 .68 gray

 

l] Occasional infrequent spots were not tabulated if RF values were
too variable to be confident of average.
3] Relative migration in relation to solvent front-~RF-1 for first
direction, RF-Z for second direction.
2] See text, pages 40-41 for description of development procedure.

 

89

Table A-8 (Cont'd.)

 

Spot :
no.

1

2
3
4

\DQN

10

ll

12
13
14

15
16
17

18
19
20
21
22
23
24

Colorgl

 

Visible (or UV)_4f

: Diaz. sulf.: NaOH

gray-tan
brown

-yel-grn

(UV)
plum purple
peach lavender
yellow
pink

v. 1t. yel.
yellow tan
pink lavender
yellow

Remarks

Runs together with 2, 3, 36, 50, and 51
along upper margin.

Large spot; overlaps 52 and 55 on g. nigra
papers.
Blurs into 57 on J, nigra papers.

Sometimes abuts 6 or 40.

Sometimes abuts 13, 16, 17 or 82; often a
large spot with irregular margin.

Hydrojuglone glucoside? Overlaps 43;
sometimes abuts 11, 13, 41.

Sometimes abuts 10, 14, 40 or 81; irregu-
lar upper margin.

Oblong spot.

Small spot.

Overlaps 11 on g, regia papers; blurs into
43 on J. nigra papers.

Overlaps 9 on g, regia papers.

Overlaps 83 on g, nigra papers.
Abuts 29, blurs into 64 on J. nigra papers.

Occasionally with irregular lower margin;
sometimes abuts 17, 45.

 

g] Visible under ultraviolet light only after reagent spray.

 

90

Table A-8 (Cont'd.)

 

 

 

 

: J. regia : J. nigra : J. regia x nigra : Colori/
: 2l: : : : : ' UV (or visible)
Spot: R -1_ :R -2:R -1 : -2: R -l : R -2 :
no.: F : F : F : RP : F : F Initial : NH3
25 .44 .68 gray
26 .52 .65 .56 .61 .56 .70 gray or
blue
27 .72 .63 blue ultra.
28 .49 .79 .48 .80 .53 .80 rose
29 .67 .78 .70 .72 .65 .75 blue gray
3O .81 .69
31 .33 .72 .34 .77 blue gray
32 .46 .79 .47 .80 ultra.
33 .59 .78 orange 1t. pink
34 .62 .73 .61 .75 blue
35 .82 .87 .80 .92 faint
36 .31 .00 .27 .00 tan
37 .65 .11 light
38 .61 .81 1t. ten
40 .63 .33 .63 .28 .54 .30 tan gray
41 .55 .41 .56 .28 .49 .23 grn-gra vis. yellow
42 .41 .43 peach
43 .52 .54 .53 .49 .53 .46 purple vis. tan
45 .50 .61 .53 .66 gray blue-gray
48 .11 .09 .07 .10 lavender
49 .29 .14 .30 .10 peach
50 .16 .00 .15 .00 brt. blue
51 .38 .00 .46 .00 .42 .00 gold, vis. green
vis. yel.
52 .46 .05 .45 .04 gray
53 .15 .08 .16 .07 blu-gra
54 .24 .11 .24 .09 via. v.
1t. yel.,
brt. blu-

green

 

 

91

Table A-8 (Cont'd.)

 

Coloilf k_
Visible (pr UV)ir

 

 

Spot : . .
no. : Diaz. sulf. : NaOH : Remarks

25 1t. yel.
26 yellow tan

27

28

29 yellow tan Abuts 20 on.g, nigra papers.
30 pink Fades away.

31

32 Overlaps 86 on hybrid papers.
33

34

35 yellow tan Blurs into solvent front.

36 gray-tan

37

38 tan Trails into solvent front.
40 yellow Sometimes abuts 11 on J. regia papers.
41

42

43

45

48

49

SO gray

51 tan Overruns 52.

52 plum lavender Oblong spot; overlaps 5; overrun by 51.
53
54

 

 

92

Table A-8 (Cont'd.)

 

 

 

 

: J. regia : J. nigra : J. regia x nigra : Colorgl
: Zl: : : : : UV jot visible)
Spot: R -1_ : -2:R -l : R -2: R -1 -2 :
no.: F :RF : F : F : F RF Initial : NH3
55 .50 .11
56 .37 .16
57 .48 .20 .47 .17 brown
58 .40 .26 .40 .21 .41 .20 ultra.
59 .23 .21 .26 .19 gray
60 .28 .34 .33 .25 .40 .24 pink to
cream

61 .34 .28 .35 .30
62 .42 .32 gray
63 .26 .28 .29 .27 purple
64 .79 .53 .73 .57 blue blu-gray
65 .35 .21 blue
66 .46 .14 faint
67 .35 .27 .31 .16 .34 .13 pink
68 .68 .42 blue
69 .15 .32 light
71 .28 .42 .33 .39 1av.-gra.
73 .52 .95 light
75 .36 .56 .30 .59 gray
76 .07 .57 dark
77 .12 .56 dark
80 .17 .22 .21 .31 ivory
81 .63 .35 blue-gray
82 .29 .35 .36 .35 via. yel.
83 .53 .53 .52 .54 grn.-gray
85 .52 .31 gray
86 .45 .79
87 .46 .67 gray
88 .50 .27 tan

. vis. yel.
89 .24 .20 dark

 

 

93

Table A-8 (Cont'd.)

 

 

 

 

Colorzl
Visible gor UV1£f

Spot : :

no. : Diaz. sulf.: NaOH Remarks

55 pink plum Small oblong spot; overlaps 5.

56 1t. tan

57 orange Often overlaps 6.

58

59 lav.-gra. Irregular lower margin.

60 yellow Sometimes abuts 61 in g, nigra papers.
61 tan Sometimes abuts 60 in J, nigra papers.
62

63

64

65

66

67 Small spot.

68

69

71

73

75 Abuts 22, 23, 24 on hybrid papers.
76 Oblong spot.

77 Small spot.

80

81 Abuts 11.

82 gray Sometimes abuts 9 on hybrid papers.
83 Overlaps 19 on J. nigra papers.

85

86 yellow Overlaps 32.

87 yellow Yellow fades away.

88 tan

89

 

 

Table A—9.-Relative frequency of occurrence of 79 spots on paper chromatograms prepared

is x nigra hybrids

and 1. re

 

from leaf extracts of Juglans regia, J. nigra,

 

Chromatogram number

J.

‘

ni ra

J.

re is

3}

 

regia x nigra

 

 

'baig
nods
mns
91*1L
ST-IL
71-1l
{I‘ll
ZT-IL
II-Il
OT-IL
6-Tl
-baa;
nods

9Z-0l
SZ-OL
VZ-OL
EZ-OL
ZZ-OL
02-01
02-04
6I-OL
SI-OL
LT-OL
9I-Ol
SI-OL
OI-OL

6-OL

I-OL
'bOl;

nods

8-IL
tilt
9-11
9-11
C-IL

 

l 1 l l l 1 1 1 8 1.00

l 1 l l 1 1 l 1 l l l 1 l l l 15 1.00
0 1 l l 0 O l O 0 O l 1 1 0 1 8
l l l 0 O 0 O 0 l 0 l l l 0 0 7

1 151 1 1 1 1 1 1 1 9 1.00
2 1 1 0 0 0 0 0 0 0 2
3 1 1 o 0 o 0 0 0 0 2

0 l l 0 0 l O 0 3 .38

.53

.22

0 0 O 0 0 O 0 0 0 0

47
.67

.22

4 l 1 l 1 l 1 l 1 l 9 1.00
5 1 l 1 l 1 l l l 1 9 1.00

l 1 l 1 l l 1 l 8 1.00
0 0 0 O 0 0 O 0 O 0

O 0 0 0 l 1 0 O 2

0 0 0 0 0 l 0 0 1

1 l l 0 l l 0 1 l l 0 0 l 1 0 10
l l 1 l 1 1 0 1 l 1 l l l l 1 l4
1 l l l l l l l l 1 0 l l l l 14
l 1 l 0 0 0 O l 0 0 l 0 0 0 l 6

.93

.25

93
.40

.44

7 l 1 l l 1 1 1 l 1 9 1.00

6 l l 0 0 0 O 0 l 1 4

94

.12

l 1 l l 0 0 0 O 4 .50

.87

l 1 0 1 1 l 0 l l l l l l l 1 13
1 l l 1 l 1 0 1 0 0 l 1 1 0 l 11

.56

8 l l l 1 0 0 O 0 l 5
9 1 0 l l l l 1 1 1 8

8 1.00
l l l 1 l l l 1 8 1.00

l l l l 1 l 1 l

.73

.89

10 l 1 1 l 1 l 1 l l 9 1.00

l l l l l l 1 l l l l l l l l 15 1.00

.80 l l l l l 1 l l 8 1.00

l l l l 1 l l 0 O O 1 l l l l 12
0 0 0 0 0 0 0 O O O l 0 O l O 2

l 1 l 1 l 1 1 1 l 9 1.00
12 l 1 l l l 1 1 1 l 9 1.00
13 l 1 l 1 l l 1 1 l 9 1.00

14 l l 0 O 0 l l 1 1 6

ll

0 0 l l 1 l l O 5 .62

0 0 1 l 0 0 0 0 2

.13

.25

.67

l 0 1 1 1 0 l O l 0_l 0 l 1 1 10
0 0 0 0 1 l 0 0 l l l 0 0 O l 6

0 0 0 0 0 O 0 0 O 0
l 0 0 0 0 0 0 0 1
l O l 0 l O l O 4

.40

.67

.12

l l l 0 O 0 0 0 0 O l 0 0 O 1 5 .33

.22

15 1 l 0 0 0 0 0 O 0 2
l6 1 l l 1 0 l 1 l 1 8
17 l l l l 0 1 1 l l 8
l8 1 0 0 0 0 0 0 0 0 1
19 1 0 l l 1 1 0 l l 7

.50

.60

l l l 0 l 1 0 0 l 0 0 0 l 1 l 9
l l 1 l 1 l 0 0 0 0 0 0 O l 0 7
l l l 0 0 0 0 O 0 O 0 0 0 0 l 4

.89

l l 1 1 l l l 1 8 1.00

0 0 0 0 0 0 0 0 0 0
0 l 0 O 1 1 0 0 3

.47

.89

.27

.11

.38

l l 1 0 0 0 0 l 1 1 1 l 1 1 1 ll .73

.78

20 1 1 l 1 1 1 1 l 1 9 1.00

l 1 l 1 l 0 O 0 5 .62

.73

l l l l 0 0 0 1 0 l 1 l l l 1 ll

 

Table A-9 (cont'd.)

 

re is x ni ra

J.

ni ra

Chromato ram number

 

 

“i“.

 

 

 

.88
.25

l l l l 1 0 1 1 7
0 l 0 0 0 l 0 0 2
0 0 l l 1 l l 0 5
0 1 0 0 O l l l 4
0 0 0 l 0 0 l 0 2
0 0 0 0 0 0 0 l 1

.93

l l 1 l l 1 l l 1 l 1 l l 1 0 l4
1 1 l 0 l 0 l l l l 0 l l l 0 ll
1 0 1 0 l 1 l l 1 0 0 0 1 1 O 9
l l 0 l l 1 1 1 0 l 0 l 1 l 0 ll
1 O 0 0 0 0 0 0 0 0 0 0 0 0 0 1
l 0 0 0 1 0 0 0 0 0 0 0 0 0 0 2
l 0 0 1 1 0 0 0 O 0 0 l 0 0 1 5

.ll
.22

21 l 0 0 0 0 0 0 0 0 l
22 1 0 0 l 0 0 0 0 0 2
23 l 1 0 O 0 l 0 0 0 3
24 1 l 1 0 l l 1 l 1 8

.73
.60

.62

.33

.50

73
.07

.89

.25

25 0 0 0 0 O 0 0 0 0 0 0
26 l l 0 1 0 1 l l 1 7

.12

13
33
.07

.78

0 0 0 0 0 0 0 0 0 0
1 1 l l l l 1 0 7

.11

27 1 0 0 0 0 0 0 0 0 l
28 l 0 0 O l 1 1 1 l 6

.88

0 0 0 0 0 0 l 0 0 0 0 0 0 0 0 l

.67

95

l l 1 l 0 0 0 0 4 .50

1 l 1 0 0 0 0 0 0 0 1 0 0 1 0 5 .33

.44

29- 1 1 0 0 O l 0 O l 4
30 l l 0 0 0 0 0 0 0 2

0 0 0 0 0 0 0 0 0 0

0 0 0 O 0 O 0 0 0 0 0 0 0 0 0 0 0

.22

.12

l O 0 0 0 0 0 0 l
1 1 0 l O 0 0 l 4

.40

.11

31 l 0 0 0 0 0 0 0 O l
32 1 l 0 0 0 0 0 O 0 2
33 l 0 0 0 0 0 0 0 0 l
34 1 l 0 0 0 0 0 0 0 2
35 1 1 0 0 0 0 0 0 0 2
36 0 l 0 0 0 0 0 0 O l
37 l l 0 0 0 0 0 O 0 2
38 0 1 0 0 0 0 0 0 0 1
40 0 1 1 0 0 0 1 1 l 5
41 0 1 0 0 0 0 0 0 l 2

O 1 0 0 0 0 0 1 0 0 0 1.1 l 1 6

.50

53
13
47
40

1 0 0 1 O l 1 0 O 0 1 l l 0 l 8 .

.22

0 0 0 0 0 0 0 0 0 0

l 0 0 l 0 0 0 0 0 0 0 0 0 0 0 2
l l l l l l 0 O 0 0 0 0 0 0 l 7
1 l l l 0 0 1 0 0 0 1 0 0 0 0 6
l 1 1 l 1 l l 0 0 0 l 1 1 0 l 11

.11

1 0 0 1 0 0 0 0 2 .25

.22

.25

l 0 1 0 0 0 0 O 2
0 0 l 0 0 0 l 0 2
0 0 l 0 0 0 0 0 l

.22

.25

73

.11

.12

0 0 O 0 0 0 0 O 0 0 0 0 0 0 0 0 0
0 O 0 0 1 l 0 0 0 0 0 0 0 0 0 2
0 1 0 0 0 0 0 0 0 0 1 0 0 l 0 3

.22

0 0 0 0 0 0 0 0 0 0
0 1 1 0 0 0 0 l 3

.13

.11

.38

0 0 0 0 0 0 0 0 0 0.

20
53

.56

l l l 0 0 0 0 1 O l l 1 0 0 1 8 .

.22

 

Table A-9 (cont'd.)

 

Chromato ram number

re is x ni ra

J.

 

n1 re

:

re’ia

 

 

 

0 0 0 0 0 0 0 O 0 0
l 1 l l 0 0 0 O 4

20

O

0 l 1 0 0 0 0 0 0 0 0 0 0 0 1 3

42 0 0 0 0 0 0 0 O 0 0 0
43 0 l 1 0 0 0 0 0 1 3
45 O O 0 O 0 0 0 0 O 0 0
48 0 0 0 l 0 0 0 0 0 1

.50
.62

l l 1 l 1 1 l 1 1 l 1 l 1 l l 15 1.00
l l l 0 l 1 0 0 0 0 0 l 0 l 0 7
0 0 0 0 0 0 l 0 0 0 0 0 0 0 0 1

.33

1 1 1 0 l 0 1 0 5

47
07
27

0 0 0 0 0 O 0 0 0 0
O 0 0 O 0 0 0 0 0 0
l 1 l 1 0 l 1 l 7

11
.22

0 0 0 1 0 l 0 0 0 0 0 1 1 0 0 4

49 0 0 0 0 0 1 l 0 O 2

.88

1 l 1 1 1 l 1 1 8 1.00

1 1 1 l 0 0 0 1 5

1 1 1 l 1 l l 1 l l 1 l 1 1 1 15 1.00
0 l l l l l 1 1 1 1 l 1 1 1 1 l4
1 1 l l 0 1 l 1 1 1 l 1 1 l l 14
1 l 1 l 1 1 1 0 1 l 0 l l 1 1 13

50 0 0 0 0 0 0 0 0 O 0 0
51 0 0 1 l 0 0 0 0 0 2

.93

.22'

96

.62

93
87

1 l 1 1 1 l l l l 1 l 1 1 l 1 15 1.00

52 0 0 0 0 0 0 0 0 0 0 0
53 0 0 l 1 0 0 0 0 0 2
' 54 0 0 0 0 0 0 0 O O 0 0

l 1 0 1 0 0 0 O 3 .38

l l 1 l 0 0 1 l 6

.22

.75

0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0

.67

l 1 1 0 0 0 0 1 l 1 l l l 1 0 10
1 1 l 0 0 0 0 0 0 0 O 0 0 0 0 3
l l l 0 l l 0 0 0 0 0 0 0 0 0 5
l l l 1 1 l 0 0 1'1 1 l 1 0 1 12
l 1 l l l 1 1 0 1 l 0 1 1 l l 13
l l l l 0 0 l 1 l l 1 0 l 1 l 12
l 0 0 O 0 0 0 l 0 1 1 l 0 1 0 6

55 O 0 O 0 0 0 0 O 0 0 0
56 0 0 0 0 0 0 0 O 0 0 0
57 0 0 0 0 0 0 0 0 0 0 0
58 0 0 1 l O l 0 l 0 4
59 0 0 0 0 0.0 0 0 0 0 0
60 0 0 1 l 0 l 0 l 0 4

20
.33

l l 1 l l 1 1 1 8 1.00

l l 0 l 0 0 0 O 3

.38

80
.87

.44

.50

1 1 1 1 0 0 O 0 4
0 l 1 1 1 0 0 0 4

.50

80
.40

.44

.25

0 1 1 O 0 0 0 O 2

61 0 0 0 0 0 0 0 O 0 0 0
62 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0
1 0 0 0 0 0 0 0 1

13
.67

0 0 l 0 l 0 0 0 0 0 O 0 0 0 0 2
l l 0 l l l 1 0 0.1 0 l l l 0 10
1 l l l 0 0 1 0 0 0 l 0 0 0 l 7

.12

63 0 0 O 0 0 0 0 0 0 0 O
64 0 1 0 0 O 0 0 0 1 2

0 0 0 0 0 0 0 0 0 0

47

.22

Table A-9 (cont'd.)

 

number
nigra

Chromatqggam

J. re is x nigra

'bal;
nods

9I-IL
ST-IL
VI’IL
EI-IL
ZI*I£
TI-Il
OI-TL

6‘11

 

'baJ;
nods

mns

9z-01
sz-oz
vz-oz
cz—oz
ZZ-OL
Roz-oz
hoz-oz

 

r4: 61-OL

re is

J.

 

O.

8I-0L
LI“0£
9I-OL
ST-OL
01-0L

6-0l

T-OL

°b313
nods
umS
8-IL
L-IL
9-IL
S-IL
E-Tl
Z-TL
I-Tl
98
34.

 

Spot
no.

 

0 0 0 0 0 0 0 0 0 0

.40

0 0 0 0 0 0 0 l 0 0 l 1 l 1 1 6

65 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 O 0 O
l 0 0 0 0 0 0 0 1

.13

1 0 0 0 O 0 0 0 0 0 O 0 0 0 1 2

66 0 0 0 0 0 0 0 0 0 0 0
67 0 0 1 0 0 l l l 0 4

.12

20
07

0 0 0 0 0 0 0 0 0 0 0 1 l 0 1 3 .

.44

0 0 0 0 0 0 0 0 0 0

l O 0 0 0 0 O 0 0 0 0 0 0 0 0 1

68 0 0 0 0 0 0 0 0 0 0 0
69 0 0 0 0 0 0 0 0 0 0 0

71 0 0 0 0 0 0 0 O 0 0 0

97

0 0 0 0 0 0 0 0 0 0
l l 0 l l l 1 1 7

13
.20

l 0 l 0 0 0 0 0 0 0 0 O 0 0 0 2

73 0 0 O 0 0 0 0 0 O 0 0
75 0 O 0 0 0 0 0 0 0 0 O

.88

1 0 0 0 0 0 0 0 0 0 l 0 0 0 l 3

.50

l 1 l l O 0 0 0 4

0 0 O 0 0 0 0 0 0 0 0 O 0 0 0 O 0

76 0 0 0 0 0 0 0 0 0 0 O

.38

l l 1 0 0 0 0 0 3
1 l l l 1 l 0 l 7

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 O
0 l l 0 0 0 1 0 0 0 0 0 1 0 O 4
0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1

77 O 0 0 0 0 0 0 0 0 0 0

.88

27
.07

80 0 0 0 0 0 0 0 0 0 0 0

.38

l 1 l 0 0 0 0 0 3
0 l 1 1 0 1 0 0 4
0 0 1 O 0 0 0 0 1
1 l l 0 0 0 0 l 4

81 0 0 0 0 0 0 0 0 0 0 0
82 0 0 0 l 0 0 0 0 0 l

.50

0 1 1 1 0 0 1 1 1 1 0 0 l l 0 9 .60

.11

.12

.47

l 1 0 0 0 0 0 1 0 O 1 1 0 l l 7

83 0 0 0 0 0 0 0 0 0 0 0
85 0 0 0 0 0 0 0 0 0 0 O

.50

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 l 0 1

l 1 1 l l 0 l 1 7 .88

07

86 0 0 0 0 0 0 0 0 0 0 O

.62

l 0 1 l l l 0 0 5
1 l 1 1 l 0 1 1 7
0 l 1 0 0 0 0 0 2

0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 0 0

87 0 0 0 0 0 0.0 0 0 0 0

.88
.25

0 0 0 0 0 0 0 0 0 0 0 0 0 0 O 0 0

88 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

89 0 O 0 0 0 0 0 0 0 0 0

 

pot was present on listed chromatograms; 0 indicates spot was absent.

1/ 1 indicates that s

 

98

Table A-10.--Spot frequency difference (s.f.d.) values for 79 spots

found on paper chromatograms prepared from leaf extracts of

Juglans nigra, J, regia, and J, regia x nigra hybrids

 

: Spot frequency difference :Spot frequency difference

 

 

 

Spot : nigra— nigra- hybrid- Spot : nigra— : nigra— : hybrid-
no. : regia : hybrid : regia : no. : regia : hybrid : regia
1 O O 0 42 .20 .20 O
2 '.31 .15 .16 43 .67 .50 .17
3 .25 .47 -.22 45 .47 -.15 .62
4 -.33 -.33 O 48 —.O4 .07 -.ll
5 -.07 .93 -1.00 49 .05 .27 -.22
6 .49 .68 -.19 50 1.00 .12 .88
7 -.6O .28 -.88 51 .71 -.O7 .78
8 .31 .37 -.O6 52 .93 .31 .62
9 -.16 -.27 .11 53 .65 .49 .16
10 0 O 0 54 1.00 .25 .75
ll -.20 -.20 O 55 .67 .67 O
12- -.87 -.49 -.38 56 .20 .20 0
13 -.27 .48 -.75 57 .33 -.67 1.00
14 -.27 .40 -.67 58 .36 .42 -.O6
15 .11 .21 -.10 59 .87 .37 .50
16 -.29 .10 -.39 6O .36 .30 .06
17 -.42 -.53 .11 61 .40 .15 .25
18 .16 .27 -.11 62 .13 .13 0
19 -.05 .35 -.4O 63 .67 .55 .12
20 -.27 .11 -.38 64 .25 .47 -.22
21 .82 .05 .77 65 .40 .40 O
22 .51 .48 .03 66 .13 .13 O
23 .27 -.02 .29 67 -.24 .08 -.32
24 -.16 .23 -.39 68 .07 .07 O
25 .07 -.18 .25 69 O -.75 .75
26 -.65 .Ol -.66 .71 .53 -.O9 .62
27 .22 .33 -.11 73 .13 .13 O
28 -.6O -.81 .21 75 .20 -.68 .88
29 -.11 -.17 .06 76 O -.50 .50
3O -.22 0 -.22 77 O -.38 .38
31 .29 .28 .Ol 80 .27 -.61 .88
32 .31 .03 .28 81 .07 -.31 .38
33 .02 .13 -.11 82 .49 .10 .39
34 .25 .22 .03 83 .47 .35 .12
35 .18 .15 .03 85 0 -.50 .50
36 .62 .48 .14 86 .07 —.81 .88
37 -.22‘ -.12 -.10 87 0 -.62 .62
38 .02 .13 -.11 88 0 -.88 .88
40 —.36 -.18 -.18 89 O -.25 .25

41 .31 .53 -.22

 

 

 

 

 

 

 

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