Comparison of selection indices to screen lowland rice for drought resistance

Summary Although many selection indices have been used to screen rices (Oryza sativa L.) for drought resistance, there has been little comparison of the relative merits of these indices. Research was conducted to compare drought resistance as estimated from grain yields, canopy-temperature-based stress indices, visual scoring, and uprooting force for 30 rice genotypes grown in the field with a puddled Maahas clay (Typic Tropaquept) and to evaluate traits related to drought resistance from nonstressed plants grown in the field and in aeroponic culture. Water deficit was imposed in the field by withholding irrigation from 45 to 75 days after transplanting compared to a continuously flooded control. Grain yields in the stress treatment were most strongly correlated with visual assessment of drought stress symptoms according to a standard evaluation system (r = 0.66). Canopy-temperature-based indices were also significantly correlated with grain yields of the stress treatment (r from –0.55 to –0.63). No trait of aeroponically grown plants was correlated with traits of stressed plants in the field. We conclude that visual scoring of stressed plants was the best method of screening for drought resistance, but if controlled water deficit cannot be imposed, then drought resistance may be estimated by measuring both uprooting force and grain yield.     

Evaluation of a simple technique for characterizing rice root systems in relation to drought resistance

Summary Drought is a constraint to rice yields in much of Asia's rainfed rice crop. Although genotypic variation for root system characteristics is well documented in this semiaquatic species, little improvement of rice for drought resistance has been attempted. No simple method to evaluate root systems exists. In this paper we report the development and testing of a simple device to measure the pulling force required to uproot rice seedlings. The force required to pull rice seedlings from the paddy soil was correlated with root weight, root branching and thick root number. The capability to separate rice cultivars by this technique and relationship of the root pulling force to other drought screening methods are illustrated.     

Transpiration response of two bread wheat lines differing in drought resilience and their backcross parent under dry-down conditions

Improving wheat productivity in drylands largely depends on how plants manage limited water resources. Using fraction of transpirable soil water threshold (FTSW_Th) and drought stress response function, we characterized the water conservation traits of two wheat multiple synthetic derivative lines (MSD53 and MSD345) which both contain introgressed segments from Aegilops tauschii but differ in drought resilience. The lines and their backcross parent, ‘Norin 61’, were subjected to dry-down conditions. MSD53 had a higher FTSW_Th for transpiration decrease than ‘Norin 61’ and MSD345. In terms of drought stress response function, MSD53 had the lowest threshold suction, suggesting a lower drought resilience capacity compared with MSD345. However, MSD53 exhibited an effective-water-use trait whereas MSD345 exhibited a water-saving trait under dry-down conditions. These results are consistent with the reported higher yield of MSD53 in comparison with MSD345 under drought stress in Sudan, and demonstrate that high FTSW_Th supports effective water use for improved agricultural productivity in drylands. The differences in water conservation traits between the two MSD lines may be attributed to variation in introgressed segments, which can be further explored for drought resilience breeding.     

Varietal differences in perennial ryegrass for nitrogen use efficiency in leaf growth following defoliation: performance in flowing solution culture and its relationship to yield under simulated grazing in the field

Varietal differences in rates of NO₃ uptake and remobilization of nitrogen (N) during a cycle of severe defoliation and regrowth were assessed in perennial ryegrass (Lolium perenne L.) varieties Ba11778, Aberelan, Talbot and Gator. Plants were grown in flowing solution culture for 34 days, prior to a 30 d treatment period, including 21 d of regrowth. Net uptake of NO₃ was measured continuously from solutions maintained automatically at 10 mmol m^-3NO₃.¹⁵ N-labelled NO_{3 3} was supplied to the plants in situ for either 21 h immediately prior to defoliation, or for 7-11 days afterwards, to measure remobilization of N from roots and stubble into the regrowing leaves. The four varieties differed only slightly in total dry matter production over 30 d, but varied substantially in post-defoliation specific growth rate by the leaf fraction. Gator was the lowest ( 0.11 g g^-1 d^-1), Ba11778 the highest ( 0.15 g g^-1 d^-1) and Aberelan and Talbot intermediate in leaf regrowth rate. Varieties did not vary significantly in shoot: root dry weight ratio, but the rate of NO₃ uptake per unit of leaf fresh weight during regrowth was 27% lower in Ba11778 than in Gator. Leaves of Ba11778 and Aberelan had a lower organic N concentration in the dry matter at the end of the regrowth period than Talbot, which, in turn, had a lower concentration than Gator. Varieties differed in amount of leaf dry matter produced per unit of N absorbed by the plants after defoliation, the values (g g^-1 N) being Ba11778 (22.6 ), Aberelan (20.7), Talbot (18.3) and Gator (16.7). More N was remobilized from the roots than from the stubble after defoliation. There were no significant differences among varieties in the amount and rate of N remobilization following defoliation. In earlier field experiments employing frequent cutting to simulate grazing, Ba11778 and Aberelan yielded at least 10% more dry matter annually than Talbot which, in turn, outyielded the amenity variety Gator by a similar amount. Thus the present results are consistent with the hypothesis that the efficiency of N use in leaf growth is an important factor determining genetic variation in productivity under frequent defoliation in the field when the herbage produced consists primarily of leaf. This revised version was published online in July 2006 with corrections to the Cover Date.     

Trait identification and QTL validation for reproductive stage drought resistance in rice using selective genotyping of near flowering RILs

Drought resistance is becoming an indispensable character for rice improvement due to the dwindling global water resources. Genetic improvement for drought resistance is achieved through physiological dissection and genetic analysis of independent component traits associated with crop productivity under stress. A subset mapping population of 93 near flowering recombinant inbred lines with uniform phenology was constituted for genetic analysis of reproductive stage drought resistance. The population was phenotyped for 22 physio-morphological traits under two contrasting water regimes imposed at reproductive stage. Broad sense heritabilities of morphological traits were lower under stress than irrigated. Predominant association of plant height, panicle exsertion and harvest index with grain yield were observed under stress. The sustenance of panicle exsertion through maintaining growth during moisture stress was found as a significant trait associated with the grain yield through minimizing spikelet sterility. Selective genotyping was carried out with 23 polymorphic microsatellite markers of the established target genomic regions for drought resistance. The study validated the association of a QTL region on the long arm of chromosome 1 with plant height, panicle length, panicle exsertion, biological yield and stomatal conductance under stress. This region, flanked by markers RM246 and RM315, was known to possess the semi-dwarf gene, sd-1. Role of another major interval lying between RM256 and RM149 on chromosome 8 in defining the drought resistance could be established through identification of QTLs associated with leaf rolling, panicle exsertion, plant height, panicle length, senescence and biological yield under moisture stress condition. Few other QTLs were also identified.     

Localization of a resistance gene and identification of sources of resistance to barley leaf stripe

In order to determine more precisely the location of the barley leaf stripe gene, called the ‘Vada-resistance gene’, on barley chromosome 2, 63 chromosome-doubled barley lines were tested. Using data on known chromosome 2 genetic markers, the ‘Vada-resistance gene’ was estimated to be located between the markers MSU21 and Xris45b, and at a distance of about 20% recombination from the powdery mildew resistance gene MILa. We suggest that the ‘Vada-resistance gene’ is designated Rdg1a and that all former leaf stripe resistance gene designations should be rejected. To identify possible new sources of resistance, 11 barley cultivars/lines known to possess leaf stripe resistance and originating from different parts of the world, were tested with one Danish and two Syrian isolates of the leaf stripe fungus. Three apparently genetically different sources of race-specific resistance were found. The ‘Vada-resistance’ in the cultivar ‘Golf was effective against seven out of eight isolates’ populations of the leaf stripe fungus differing in geographical origin.     

Influence of development stage and host genotype on three components of partial resistance to leaf rust in spring wheat

Summary Latency period (LP), infection frequency (IF) and urediosorus size (US) of leaf rust were determined on primary leaves and young flag leaves of 18 spring wheat cultivars. A large growth stage effect and a large cultivar effect on all three components were observed. Partial resistance as measured by the three components was generally better expressed in the adult plant stage than in the seedling stage. Associated variation of the components was observed: long LP, low IF and small US tended to go together. The association was not complete, cultivars with clear deviations of this association for one of the components were found suggesting the existence of at least partly different genetic factors controlling the respective components. LP measured on flag leaves gave the most reliable results and, therefore, could best be used as a selection criterion in breeding programs for partial resistance.     

Impact of nitrogen supply on leaf water relations and physiological traits in a set of potato (Solanum tuberosum L.) cultivars under drought stress

Improved adaptation of potato to limited water availability is needed for stable yields under drought. The maintenance of the cell water status and protection of cellular components against dehydration are important for drought tolerance, and the N status of plants affects the regulation of various respective metabolic processes. A 2‐year pot trial with 17 potato cultivars was conducted under a rain‐out shelter including two water regimes and two N‐levels to investigate genotypic differences concerning osmotic adjustment (OA) and relevant biochemical traits in relation to nitrogen (N) supply. Drought stress resulted in a rapid decrease in the leaf osmotic potential. The N, protein and proline contents increased under drought, while the N protein/N_Kjeldahl ratio decreased. Initially, total soluble sugars increased at both N‐levels but dropped back to the control level at high N‐availability under prolonged drought while remaining high in N‐deficient plants. Results indicate that potatoes have only a limited capacity of active OA and that increasing sugar and proline concentrations are rather associated with the protection of cellular components. High N supply promoted the N protein/N_Kjeldahl ratio at short‐term drought and enhanced proline accumulation. Significant genotypic differences were observed for all investigated traits.     

Molecular marker assisted tagging of morphological and physiological traits under two contrasting moisture regimes at peak vegetative stage in rice (Oryza sativa L.)

Root morphology under well-watered conditions sampled on two occasions and under low-moisture stress was studied in a randomly chosen subset of 56 doubled haploid lines derived from a cross between IR64 and Azucena at two growth stages during the vegetative stage. A molecular map of the same population served as the basis for locating QTLs controlling root morphology and associated traits. The region flanking the RFLP markers RZ730 and RZ801 on chromosome 1 were associated with plant height in all three sampling environments. This position corresponds to sd-1 a semi-dwarfing gene. A total of 15 QTL were detected at the two developmental stages, of which only three QTL were common. Region flanked by RG157 and RZ318 (chromosome 2) contained QTL for root thickness under two different developmental stages. In total, 21 QTL for different traits were detected under low-moisture stress condition. While two QTL for plant height on chromosomes 1 and 3 were common, none of the loci for root morphological traits was common between the two different moisture regimes. The chromosomal segment between RG171 and RG157 contained QTL controlling tiller number per plant, total root length, root volume and total root number per plant. Absence of common QTL for root traits between two developmental stages and two moisture regimes suggests the existence of parallel genetic pathways operating at different growth stages and moisture regimes. Root volume and total root number per plant decreased significantly under stress, whereas maximum root length and plant height exhibited non-significant increases under stress. This revised version was published online in July 2006 with corrections to the Cover Date.     

大气CO_2倍增条件下冬小麦气体交换对高温干旱及复水过程的响应

探究大气CO_2浓度倍增条件下冬小麦气体交换参数对高温干旱及复水过程的生理响应机制,有助于提高生态过程模型的模拟精度,更加准确地预测全球气候变化对农田生态系统初级生产力及其生态服务功能的影响。利用4个可精准控制CO_2浓度和温度的大型人工气候室,研究了CO_2浓度倍增条件下高温干旱及复水过程对冬小麦气孔特征和气体交换参数的影响。结果表明, CO_2浓度倍增(E)导致冬小麦远轴面气孔密度增加、气孔宽度减小、气孔空间分布规则程度降低,但提高叶片的净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)和水分利用效率(WUE)。高温干旱(HD)使叶片气孔长度、密度、周长和面积减小,导致叶片气体交换参数均显著下降。然而,高CO_2浓度及高温干旱(EHD)导致气体交换参数下降幅度相对较小,表明高CO_2浓度对高温干旱具有一定的缓解作用。此外,干旱复水后,不同处理条件下冬小麦叶片气体交换参数均有所升高,但高温干旱下叶片的气体交换参数仍未能恢复到对照水平,暗示光合器官可能在高温干旱时遭到损伤和破坏。     

Effects of a photoperiod-response gene Ppd-D1 on yield potential and drought resistance in UK winter wheat

Using a pair of near-isogenic lines(NILs) of winter wheat (Triticumaestivum L.) contrasting for the Ppd-D1 and ppd-D1 alleles, in eachof Mercia and Cappelle-Desprez, experimentsin two seasons (1997/8 and 1998/9) on aloamy medium sand examined differences inflowering date, resource capture, biomassproduction and grain yield responses toirrigation. Drought did not occur for anysustained period in unirrigated conditionsin 1998 due to high seasonal rainfall. In1999, drought developed post-floweringunder unirrigated conditions. Ppd-D1on average advanced flowering by 12 days inMercia and 9 days in Cappelle-Desprez.Earlier flowering with Ppd-D1 was dueto a shorter thermal duration from cropemergence to GS31, with no effect on thethermal duration from GS31 to GS61. In bothgenetic backgrounds, Ppd-D1 decreasedabove-ground dry matter (AGDM) at harvestin irrigated conditions by 0.3–0.9 tha^-1 (p< 0.05), but thiswas compensated for by increases inharvest index (HI), so that grain yield wasconserved. Although Ppd-D1 decreasedmaximum green area index (GAI) by 0.8–1.9this was countered by greater maintenanceof green area after flowering, so thatradiation interception during grain fillingwas conserved. The Ppd-D1 alleledecreased season-long crop water uptake inthe Mercia NILs in irrigated conditions by39 mm. Effects of drought in 1999,averaging across NILs, were todecrease machine-harvested grain yield by 0.6 t ha^-1 in Mercia and by 1.8 tha^-1 in Cappelle-Desprez (p<0.05). The Ppd-D1 and ppd-D1NILs, though, responded similarly todrought in both genetic backgrounds. Earlyflowering with Ppd-D1 decreasedpre-flowering water uptake underunirrigated conditions by ca. 25 mm,but increased post-flowering uptake by only10 mm, compared to ppd-D1. This was aresult of smaller season-long water uptakefor Ppd-D1 compared to ppd-D1.Ppd-D1 decreased stem solublecarbohydrate measured shortly afterflowering under drought by ca. 0.3 tha^-1. Effects of Ppd-D1 onother drought-resistance traits, such aswater-use efficiency (WUE; AGDM per unitcrop evapotranspiration) and maximumrooting depth, appeared to be neutral. Itis concluded that the effects of the Ppd-D1 allele appeared to be largelyneutral on yield potential and late-seasondrought resistance in the UK's temperateenvironment in these genetic backgrounds.However, there were indications that Ppd-D1 may offer scope for breeding winterwheat cultivars with more efficientproduction of grain DM per unit seasonalcrop evapotranspiration, associated withimproved HI, compared to currentlycommercial UK genotypes.     

Influence of race and post infection temperature on two components of partial resistance to wheat leaf rust in seedlings of wheat

Summary Components of partial resistance, infection frequency and latency period, were determined in 71 winter and spring wheat genotypes in the seedling stage, after infection with three races of leaf rust (Felix 3B, Clement B and Betuwe 85C) at three different day/night temperature regimes (24/18°C, 18/12°C and 12/6°C). The genotypes were split into two groups and two separate experiments were carried out. Five genotypes, SVP 84039, Akabozu, Banco, BH 1146 and Orso, conferred a low infection frequency and a long latency period and Westphal 12A a long latency period, indicating a relatively high level of partial resistance. The correlation coefficient between infection frequency and latency period was low. Race-specificity was not found. There was a significant temperature effect on the latency period. In the second experiment the temperature x genotype interaction was significant. Temperature-response functions of transformed data demonstrated that the latency periods of four relatively resistant genotypes, Westphal 12A, Banco, BH 1146 and Orso and of Sarno and Mirela were most sensitive to temperature. The range between the genotypes with the longest and the shortest latency period was highest at 12°C. Therefore, low temperature regimes are preferred to distinguish differences in level of partial resistance.     

Partial resistance of barley to leaf rust,Puccinia hordei. IV. Effect of cultivar and development stage on infection frequency

Summary The cultivar effect on infection frequency (IF) was studied in the seedling and adult plant stages of 15 spring barley cultivars. In both stages the cultivar effects were highly significant. The cultivars L94 and Vada represented the extremes. Vada having about 2 1/2 times fewer uredosori than L94. Between cultivars and development stage clear interactions occurred. Pauline f.i. had the same low IF as Vada in the seedling stage, but in the adult plant stage its IF was about 70% higher. Also other effects could influence the cultivar effects. Increasing leaf age appears to increase IF. The cultivar effect also seemed to depend on the level of IF. At high levels the cultivars differed far less than at low levels of IF. The cultivar effect on IF appeared correlated with partial resistance in the field (r=0.7) through a high correlation with the cultivar effect on latent period (r=0.8).     

Comparison of two crossing and four selection schemes for yield, yield traits, and slow rusting resistance to leaf rust in wheat

The most important breeding objectives in crop improvement are improving grain yield, grain quality, and resistances to various biotic and abiotic stresses. The objectives of our study were to compare two crossing and four selection schemes for grain yield, yield traits, and slow rusting resistance to leaf rust (Puccinia recondita) based on additive genes in wheat (Triticum aestivum), and to identify the most efficient crossing and selection methodologies in terms of genetic gains and cost efficiency. Segregating populations were derived from 18 simple crosses and the same number of top (three-way) crosses. Half of the crosses were derived from Yecora 70 and the other half from Veery #10 as the common leaf rust susceptible parents. The four selection schemes were: pedigree, modified bulk (F₂ and F₁-top as pedigree, selected lines in F₃, F₄, F₂-top, F₃-top as bulk; and pedigree in F₅ and F₄-top populations), selected bulk (selected plants in F₂, F₃, F₄, F₁-top, F₂-top and F₃-top as bulk; and pedigree in F₅ and F₄-top populations), and nonselected bulk (bulk in F₂, F₃, F₄, F₁-top, F₂-top and F₃-top; and pedigree in F₅ and F₄-top populations). A total of 320 progeny lines, parents and checks were tested for grain yield, other agronomic traits and leaf rust resistance during the 1992/93 and 1993/94 seasons in Ciudad Obregon (Sonora State, Mexico) which represents a typical high yielding irrigated site. The influence of the type of cross and the selection scheme on the mean grain yield and other traits of the progenies was minimal. The selection of parents was the most important feature in imparting yield potential and other favourable agronomic traits. Moreover, the highest yielding lines were distributed equally. Progeny lines derived from Veery #10 crosses had significantly higher mean grain yield compared to those derived from the Yecora 70 crosses. Furthermore, a large proportion of the highest yielding lines also originated from Veery #10 crosses. Mean leaf rust severity of the top cross progenies was lower than that of the simple cross progenies possibly because two parents contributed resistance to top cross progenies. Mean leaf rust severity of the nonselected bulk derivatives was twice that of lines derived from the other three schemes. Selected bulk appears to be the most attractive selection scheme in terms of genetic gains and cost efficiency. This revised version was published online in August 2006 with corrections to the Cover Date.     

Level of partial resistance to leaf rust,Puccinia hordei,in West-European barley and how to select for it

Summary The partial resistance to leaf rust (Puccinia hordei) of 40 West-European spring barley cultivars was measured in plots isolated from one another to reduce inter plot interference. The leaf area affected by leaf rust was also measured in small plots of 0.5 m² adjacent to each other, and on individual plants. The latent period was measured in the seedling stage and the adult plant stage, the infection frequency in the seedling stage only. The cultivars varied widely for partial resistance, many cultivars carrying a considerable level. Both the small adjacent plots and the single plants showed a marked inter plot interference strongly reducing the difference between cultivars. H wever, the ranking order of the cultivars was hardly, if at all, affected. Both latent period and the infection frequency showed large differences between cultivars, the latent period in the adult plant stage being highly correlated (r=0.82) with partial resistance, infection frequency in the seedling stage only rather weakly (r=–0.33).Selection for partial resistance appeared very effective in all stages tested; the seedling, the single adult plant, and the small plot stage. Selection in the small plot stage was the most effective followed by selection in the seedling stage. Selection for partial resistance therefore appears very well possible at all stages of the selection program.     

Pleiotropic association of infection frequency and latent period of two barley cultivars partially resistant to barley leaf rust

Summary Cebada Capa, carrying four to six minor genes for a longer latent period (LP), was crossed to L94 and Vada, carrying no and five to six minor genes for a longer LP respectively. Of each of 68 F3-lines the infection frequency (IF) and the LP of ten just-heading plants were assessed. There appeared to be a strong association between IF and LP, whereby the relationship between IF and LP of both crosses could be described by a single linear regression equation. The data strongly suggest that the genes for increased LP pleiotropically decrease the IF. The possibility of a close linkage between genes for reduced IF and genes for increased LP, although unlikely, could not be excluded.     

Functional mechanisms of drought tolerance in maize through phenotyping and genotyping under well watered and water stressed conditions

Developing tolerant genotypes is crucial for stabilizing maize productivity under drought stress conditions as it is one of the most important abiotic stresses affecting crop yields. Twenty seven genotypes of maize (Zea mays L.) were evaluated for drought tolerance for three seasons under well watered and water stressed conditions to identify interactions amongst various tolerance traits and grain yield as well as their association with SSR markers. The study revealed considerable genetic diversity and significant variations for genotypes, environment and genotype × environment interactions for all the traits. The ranking of genotypes based on drought susceptibility index for morpho-physiological traits was similar to that based on grain yield and principal component analysis. Analysis of trait – trait and trait – yield associations indicated significant positive correlations amongst the water relations traits of relative water content (RWC), leaf water potential and osmotic potential as well as of RWC with grain yield under water stressed condition. Molecular analysis using 40 SSRs revealed 32 as polymorphic and 62 unique alleles were detected across 27 genotypes. Cluster analysis resulted in categorization of the genotypes into five distinct groups which was similar to that using principal component analysis. Based on overall performance across seasons tolerant and susceptible genotypes were identified for eventual utilization in breeding programs as well as for QTL identification. The marker-trait association analysis revealed significant associations between few SSR markers with water relations as well as yield contributing traits under water stressed conditions. These associations highlight the importance of functional mechanisms of intrinsic tolerance and cumulative traits for drought tolerance in maize.     

Ability of chromosome 4H to compensate for 4D in response to drought stress in a newly developed and identified wheat–barley 4H(4D) disomic substitution line

A spontaneously developed wheat–barley 4H(4D) disomic substitution line was identified cytogenetically using genomic in situ hybridization (GISH), multicolour fluorescent in situ hybridization (FISH) and microsatellite markers. The ability of the barley 4H chromosome to compensate for wheat 4D in response to mild drought stress was also investigated. In the barley cv. 'Betzes' and the 4H(4D) substitution line, mild osmotic stress induced intensive stomatal closure, resulting in reduced water loss through transpiration and unchanged relative water content in the leaves. As the CO₂ assimilation rate remained relatively high, the water use efficiency, which is an important factor associated with drought tolerance, increased extensively under mild osmotic stress in these lines. In the case of the parental wheat genotypes, however, mild drought stress induced less intense stomatal closure and a greater decrease in the CO₂ assimilation rate than in barley or in the substitution line, resulting in unaugmented or reduced water use efficiency. The results demonstrate that genes localised on the 4H chromosome of barley were able to increase the water use efficiency of the wheat substitution line, which is suitable for improving wheat drought tolerance through intergeneric crossing.     

Quantity production of anther-derived haploids from a multiple disease resistant tobacco hybrid. I. Frequency of plants with resistance or susceptibility to tobacco mosaic virus (TMV), potato virus Y (PVY), and root knot (RK)

Summary Methods are described for producing large numbers of haploid plantlets from anthers of a flue-cured tobacco hybrid with monogenic resistance to tobacco mosaic virus, (TMV), potato virus Y (PVY) and root knot (RK), respectively. Additional details are given on colchicine treatment for converting haploids to doubled haploids (DH's) and on the frequency of spontaneous DH's among untreated plantlets. Disparate genetic ratios of TMV-resistant to TMV-susceptible plants were obtained among colchicine-treated haploid plantlets, induced DH's and untreated haploids when compared with F₂ and BC₁ progenies. Haploids (gametes) with the gene for TMV resistance occurred more frequently than expected and plantlets with the gene for RK resistance occurred less frequenctly than expected. Transmission of the gene for PVY resistance differed only slightly from Mendelian expectations. These unexpected ratios, in addition to the frequent occurrence of plastid chimeras among anther-derived plantlets, strengthened our conviction that haploidy is somehow associated with mutation.Joint contribution from the Departments of Genetics, Crop Science and Plant Pathology, North Carolina State University, and the Tobacco Research Laboratory, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Oxford, North Carolina. Paper No. 5576 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina.