HONOLULU, HAWAII

University of Hawaii

Near isogenic lines (NIL) of inbred Hi27

--James L. Brewbaker and Aleksander D. Josue

The Hi27 NIL series was initiated in 1967 to provide tropically-adapted mutants to scientists working in the tropics (MGN 42:37-38). Each mutant was to be backcrossed at least six times to Hi27, a hardy tropical flint inbred that we selfed out of inbred CM104, created in India as a sib-line from the Colombian flint Amarillo Theobromina (pedigree = A Theo 21-B-6#-15-7#). Hi27 generally tolerates most tropical diseases and environmental insults, and is homozygous for loci such as A1 A2 b Bz C-I Mv p-ww Pl pr r Y. In 1995 we published a list of mutant loci that had been entered through backcrossing (MGN 69:58-59). All of the mutants listed at that time have now been backcrossed six or more times to Hi27, with the following changes:

(1) Mutants that could not be maintained from the 1995 list:

bk, bt2, lc, mn, pg2, rf, rt, v2 and w3

(2) Mutants that have been added to this list:

a2, bk2, bt1-A, c2, j2, ms6, ms8, o5, Tlr, y8 and y11

(3) New mutants (temporary symbols) under study:

blo (blotch), bst (brown-stripe), dcb (double-cob), lc2 (leaf-color), lfl (leaf-fleck), nl3 (narrow-leaf), os (opaque-small), sky (skinny) and zb232 (zebra)

The complete NIL set now includes 97 mapped genes and the 9 mutants under study. More than 200 genotypes are now available, including many digenic and multi-genic combinations such as bm3 gt and C sh bz wx. All are being provided to the Maize Genetics Coop, e.g., symbolized sh2^Hi27.

Grassy Tiller and Sweet Corn

---Brewbaker, James L.

Tillering is a characteristic of early American sweet corns (sugary-1), but is rarely found in other races or types of maize. We report here that all of the early sweet corns we've tested carry the gene, grassy tiller (gt). Grassy-tillered plants also produce leaves that extend the husks ("husk leaves" or "flag leaves"), a feature not noted in the genetic literature but of utility to processors of temperate sweet corn for removal of husks.

Our breeding of tropical sweet corns in Hawaii has been based entirely on hybrids of temperate (tillered) and tropical (non-tillered) types. All of the >20 open-pollinated populations we’ve released of this type segregated tillered plants as a recessive trait (Brewbaker, HortSci 33:1262-4). Tillered plants were also marked by presence of husk leaves that segregated as a recessive monogene (MGN 79:14), now known also to be gt1.

In the present study, temperate sweet corn inbreds provided by Bill Tracy (U. Wis.) were crossed with two sources of gt, one based on population WGRComp2 from Jim Coors (U. Wis.) and one, gt^Hi27 from our near-isogenic line series (MGN 69:58-9). The temperate inbreds were:

sugary-1: 101t, C5, C40, Hotevilla AZ, P39, P51

shrunken-2: Ia453sh2

Hybrids of these sweet inbreds with gt stocks were all highly tillered, with long flag leaves (Figure 1). All F2 populations grown from these hybrids were also 100% tillered. One recombinant inbred population (SET M) based on the cross of Ia453sh2 (tillered) with Hi38bt (no tillers) segregated 19 tillered and 27 non-tillered RILs, while the F2 of this cross segregated 3:1 for normal to grassy tillered. One of the tillering NILs, M23, was crossed to a gt stock and produced only grassy-tillered hybrids.

Figure 1. Grassy tillered hybrid of NIL gt^Hi27 with sweet corn inbred P51

The number and size of tillers and husk leaves is highly correlated with plant vigor. Experimental trials at Waimanalo, Hawaii, are planted year-round, and corn biomass yields in summer are roughly double those in winter. Yields are reduced largely by low light in our wet winters (Jan. avg. 275 cal/cm^-2day^-1) vs. the dry summers (July avg. 450 cal/cm^-2day^-1). Tiller numbers are reduced in winter; the tiller heights of inbred gt^Hi27 were reduced to <6” in winter vs. >18” in summer. Vigorous +/gt hybrids often produce small flag leaves in the summer also. High plant density and low nitrogen fertility reduce the expression of tillers and flag leaves. Husk-leaf extension increased greatly in Hawaii’s summer trials for many hugely tillering Korean genotypes (MGN 59:14).

Other highly-tillering genes include Tlr (tillering) and tb (teosinte-branched), and both of these mutants also have long flag leaves. The genes are on long arm of Chromosome 1 and possibly allelic. Both genes have a major effect on ear morphology, unlike gt. The Tlr/Tlr homozygote is extremely grassy in Hawaii and has abortive ears. It resembles Cg (corngrass), a mutant that also leads to tillering and flag leaves.

Teosinte species tiller abundantly like most grasses, presumably based on genes like Tlr. Our hybrids of maize with Jutiapa teosinte and with Zea diploperennis were all highly tillered, showing tillering to be dominant (cf. Fig. 1, Srinivasan and Brewbaker, Maydica 44:353-370). In the referenced study Srinivasan produced 11 hybrids between tropical maize inbreds (with only the single main culm) and Z. diploperennis (avg. 18.3 tillers in winter, 29.3 in summer). In summer plantings the F1 plants averaged 5.4 tillers, F2’s averaged 3.4, backcrosses to maize averaged 1.6 and backcrosses to Z.d. averaged 2.9. Generation mean analysis showed that narrow-sense heritability was high (81%) and based largely on dominance and epistatic (dd) interactions. At least two loci were inferred. Winter data for the Z.d. x maize populations showed that tillers were reduced an average of 13.8% for the four generations, with similar reduction in heritability.

We have bred a broad-based population, HIC9d, from backcrosses of these Z.d. hybrids to maize. It segregates about 10% tillered plants. The population is highly heterogeneous for tiller and husk leaf extension, and for vigor, prolificacy and many ear traits. It is being tested for allelism of tillering genes to gt and Tlr.

The Maize Genetics Coop gene gt is located near centromere on Chromosome 1 and is attributed to Don Shaver (MGN 39:18-22), who writes (pers. commun.) “Earl Patterson had told me that E.G. Anderson found it or discovered it (at Cal Tech)”. The gt in our NIL set (reported in MGN 69:58-9) derives from the MGC stock gt/gt id/id (66Cal, 3327x28) that seems to have the same origin, out of mutants from Bikini in Anderson’s collection in 1948, a nursery in which I was privileged to work with Earl, Ed Coe and Andy. However, Walt Galinat (pers. commun.) notes his early interest in tillering and the possibility that the N.E. sugary lines in his program provided the gt locus of Shaver, who made hybrids of Galinat’s sweet corns with id (also found on Chromosome 1L) and pe stocks in studies of perennialism in maize (Shaver, J. Hered. 58:270-273; MGN 79:39-41). In any event, the two sources appear to be identical alleles.

In view of the rarity of tillering in maize, the independent origin of gt in early American sweet corns or their progenitors appears highly probable. Mysteriously eluding early authors on this subject was the fact that gt also controls husk-leaf extension, a feature that became of value to the temperate sweet corn industry by facilitating husk removal during processing. In Thailand the tropical supersweets with Hawaiian ancestry (many husks but no husk leaves) from 150,000 A. annually are husked following sprays with hot water (Taweesak Pulam, pers. commun.). It is unclear whether genotypes exist with flag leaves but no capacity or totipotency for tillering. We suspect that source of cytoplasm must be considered in unravelling the perennialism of Z. diploperennis that has been elusive in maize hybrids with genes like gt, Tlr, id and pe.

Heterosis among near-isogenic lines of Hi27

--Aleksander D. Josue and James L. Brewbaker

Ten mutants in our Hi27 NIL series, one on each of the 10 chromosomes, were chosen for a diallel analysis of heterosis. Each mutant had been backcrossed at least six times to Hi27, hardy tropical flint inbred (see above). Our NILs are sibbed following backcrossing, allowing preservation of some heterozygosity (1.5625% >BC6, 0.0977% >BC10). However, there is much evidence of linkage drag in such conversions, linkage that could also be associated with inter-NIL heterosis. Linkage drag with loci na1^Hi27 (3L-101) and lg2^Hi27 (3L-113) led us (Ming et al., MNL 69:60) to the Mv/mv locus on Chromosome 3L-80 (all temperate corn carries allele mv for susceptibility to the tropical maize mosaic virus). Current studies in Hawaii seek to use linkage drag in spotting other QTLs of importance to corn breeders.

It can be conjectured that QTLs for yield heterosis are often linked to mutant genes we’ve backcrossed into Hi27. To test our hypothesis, ten NIL (one per chromosome) were crossed diallely, including parent Hi27 (Griffing method 2). Mutants selected were located at 1S-55 (gt^Hi27), 2S-11 (lg^Hi27), 3L-149.0 (a^Hi27), 4S-(55) (bm3^Hi27), 5S-41 (bm^Hi27), 6L-17 (y^Hi27), 7S-16 (o2^Hi27), 8-(0) (rf4^Hi27), 9S-31 (bz^Hi27) and 10L-64 (R-nj^Hi27). Mutant rf4 had been advanced 12 backcrosses. The diallel entries were planted in single-row 5m plots in Field S1-4 at Waimanalo on May 23 and June 21, 2006. Data were taken from two samples per row of 5 plants, with months treated as replications.

Heterosis among the 53 hybrids (two were omitted due to poor stand) was universal for measured traits. Highly significant differences (P<0.001) were observed for yield (as gm. per plant), for ear length and ear diameters in cm. (Table 1), and also for plant heights (not shown). The Experimental Error interaction of NIL x “Reps” (Months) was never significant when tested against sampling error, a reflection of the homogeneity of Waimanalo soils on which our breeding nurseries have been grown since the 1960’s.

Table 1. ANOVA for Yield, Ear Length and Ear Diameter
Source	df	Yield		EL		ED
Entries	63	456.3	**	1.60	**	0.05	**
Reps	1	420.9	ns	2.41	*	0.09	*
NIL & Parent	10	277.6	*	2.04	**	0.06	**
F1s	52	375.3	**	1.10	**	0.04	**
Heterosis (NIL vs F1s)	1	6,456.4	**	22.93	**	0.39	**
EE (Ent x Rep)	63	115.7	ns	0.39	ns	0.01	ns
SE	128	584.4		3.47		0.13
^**,* - Significant at the 5% and 1% level of probability

Means and standard deviations of these three sets of data are summarized in Table 2. The NILs were similar to their parent inbred Hi27, while all hybrids were significantly higher in yield and ear traits. Relative homogeneity characterized all traits, as evident in the standard deviations and CV values.

Table 2. Means and Standard Deviations for Yield and Ear Traits
	YIELD			EL			ED
Hi27	92.5	±	16.9	13.8	±	1.4	3.95	±	0.17
NILs	93.8	±	20.4	12.7	±	1.7	4.05	±	0.32
Hybrids	121.1	±	17.8	14.4	±	1.3	4.25	±	0.27

Individual variations were seen in GCA (general combining ability) and mid-parent heterosis values, and these will be studied in greater detail following duplicate plantings in 2007. Mutant bz^Hi27 had the highest GCA for yield, but it is in a known linkage group with C, and would be expected to have greater linkage drag in our conversions to Hi27 (which is Bz C-I). GCA for hybrid yield minimized for rf4^Hi27 (97.6 gm/plant). However, the rf4 conversion represented BC12 (~ .0244% heterozygosity) and the male-sterile hybrids were grown as a block, both facts helping account for their reduced apparent heterosis for yield.

Heterosis among NIL hybrids clearly can reflect the remnant of heterozygosity among their very distinct temperate dent and tropical flint parents; indeed, we make much use of modified sister-single crosses in our supersweet tropical breeding to exploit this kind of heterosis. But it is similarly clear that linkage drag with QTLs affecting vigor and yield may play a role in this heterotic response. The dent x flint heterosis is very widely exploited in tropical corn breeding, and localization of significant QTLs may improve our genetic advance.