Variation for Heat Tolerance During Seed Germination in Diverse Carrot [Daucus carota (L.)] Germplasm

Carrot production is constrained by high levels of heat stress during the germination stage in many global regions. Few studies have been published evaluating the effect of heat stress on carrot seed germination or screening for genetic heat stress tolerance. The objectives of this study were to evaluate the response of diverse carrot germplasm to heat stress, identify heat-tolerant germplasm that may be used by plant breeders, and define the appropriate temperature for assessing heat tolerance in germinating carrot seed. To identify an appropriate screening temperature, three commercial hybrids and an open pollinated variety were evaluated at five temperatures (24, 32.5, 35, 37.5, and 40 °C). In preliminary studies, 35 °C was identified as the optimal temperature for screening heat tolerance of carrot seed. Cultivated and wild carrot plant introductions (PIs) (n = 270) from the U.S. Department of Agriculture (USDA) National Plant Germplasm System (NPGS) representing 41 countries, inbred lines from the USDA Agricultural Research Service (n = 15), and widely grown commercial hybrids (n = 8) were evaluated for heat tolerance under heat stress and nonstress conditions (35 °C and 24 °C, respectively) by calculating absolute decrease in percent germination (AD), inhibition index (II), relative heat tolerance (RHT), and heat tolerance index (HTI). All measurements of heat tolerance identified significant differences among accessions; AD ranged from −13.0% to 86.7%, II ranged from 35.7% to 100.0%, RHT ranged from 0 to 1.36, and HTI ranged from 0.0 to 1.45. The broad-sense heritability (H2) calculations ranged from 0.64 to 0.86 for different traits, indicating a moderately strong genetic contribution to the phenotypic variation. Several wild carrot accessions and inbred lines displayed low levels of heat tolerance, whereas cultivated accessions PI 643114 (United States), PI 652400 and PI 652403 (Turkey), PI 652208 (China), and PI 652403 (Russia) were most heat tolerant. This is the first evaluation of heritability for heat stress tolerance during carrot seed germination, the first measure of HTI, and the first correlation calculation between heat and salt tolerance during germination in carrot.

The ambient temperature of our planet has significantly increased over the past century (0.13°C per decade in last 50 years) as part of current global climate change attributed to anthropogenic activities and natural factors (Rohde et al., 2013). Modern climate models have predicted that mean global temperature will rise by 1.1°C by the end of this century and intense heat waves will also occur more frequently (IPCC, 2013). Heat stress, caused by elevated temperature, is a major threat for cereals, fruits, and vegetable production, especially in warm regions of the world (Farooq et al., 2011;Hasanuzzaman et al., 2013). Vegetables are a rich source of essential dietary micronutrients (Graham et al., 1999) but the effects of heat stress on the productivity of most vegetables have yet not been well-characterized (Fahad et al., 2017;Mahmood et al., 2017). It may be anticipated that cool-season vegetables, like carrot, are especially sensitive to heat stress.
Carrot (Daucus carota L., 2n = 2x = 18) is a biennial, cool-season, Apiaceous root vegetable that is a rich dietary source of provitamin A carotenoids (a-and b-carotene), which is vital for human development and health (Tanumihardjo, 2012). The optimal germination temperature of carrot is 20 to 30°C (Corbineau et al., 1994) and, like most crops, abiotic stress reduces carrot yield (Bano et al., 2014, Kahouli et al., 2014. Heat, drought, and/or salinity-induced stress contributes to the much lower carrot yields in countries like Pakistan and most of South Asia relative to North America or Europe (FAO, 2017).
Previous studies have reported that temperatures higher than 27°C can cause significant reduction in root mass, which accounts for the yield of a carrot (Rosenfeld, 2004). High temperature causes heat stress and is considered to be one of the major abiotic factors that limits carrot production because of its adverse effects on seed germination, seedling emergence, and plant growth (Landjeva et al., 2008). Heat tolerance is a complex trait that varies with the severity of stress and plant growth stage. Therefore, there is a need to identify heattolerant carrot germplasm with stable growth and yield under high temperature at various stages of growth. Seedling establishment, vegetative growth, and the reproductive phase of the life cycle in carrot are all critical developmental stages, but without the ability to germinate under heat stress, tolerance at later stages of growth is irrelevant. Heat stress tolerance in seeds is typically a multicomplex and polygenic trait (Senthil-Kumar et al., 2007) that varies from species to species and environmental conditions (intensity and exposure period of stress). It has been demonstrated in previous studies that the seed germination of various crops, including wheat (Balla et al., 2012), maize (Iloh et al., 2014;Riley, 1981), rice (Shah et al., 2011), chick pea (Sleimi et al., 2013), and spinach (Hum-Musser et al., 1999), is adversely affected as temperature increases.
Several previous studies demonstrated the negative effects of low temperature on carrot seed germination and stand establishment Pereira et al., 2007;Vieira et al., 2005), but relatively little has been reported about the effects of high temperatures. Vieira et al. (2005) demonstrated that high temperature inhibits carrot seed germination. High temperature affects some physiological and biochemical processes like oxygen requirement during seed imbibitions (Nascimento et al., 2008). There is no known mechanism for thermotolerance in carrot and insufficient information is available to characterize the influence of high temperature on carrot seed germination and biomass production. The most expansive carrot germplasm evaluation, to date, for heat tolerance at the germination stage, was done by Nascimento et al. (2008), who evaluated 34 commercial cultivars and 63 diverse germplasm accessions. They evaluated heat stress traits including ethylene production, time to first germination, and RHT under 35°C. The current lack of information limits our understanding of heat tolerance mechanisms in carrot. To expand the information needed to better understand those mechanisms, the objectives of this study were to identify the optimal temperature conditions for evaluating heat tolerance in carrot; to evaluate a large carrot germplasm collection for heat tolerance traits, including HTI, II, and AD at the germination stage; to evaluate the relationship between domestication status and root color on heat tolerance; and to calculate broad-sense heritability for carrot.

Materials and Methods
Germplasm. A total of 293 carrot genetic stocks consisting of 207 cultivated and 63 wild accessions from the USDA NPGS collection of PIs, 15 inbred lines from the USDA carrot breeding program, and eight commercial fresh market hybrids widely grown in the United States were included in this analysis. To better identify heat stress effects on germination, carrot accessions with more than 50% germination under nonstress conditions were included, as in recent evaluations of salinity tolerance (Bolton and Simon, 2019). The 293 carrot accessions originated from 41 countries and were classified into 14 regions based on their origin (Eastern Africa, Northern Africa, Southern Africa, North America, South America, Central Asia, Eastern Asia, Southern Asia, Western Asia, Eastern Europe, Northern Europe, Southern Europe, Western Europe, and Oceania) and they represent much of the global genetic diversity for carrot (Iorizzo et al., 2013). A subset of these accessions is also being evaluated in field trials in Bangladesh and Pakistan for heat tolerance at various stages of growth (A. Ali and M.A. Rahim, personal communication).
Determination of optimal heat tolerance evaluation temperature. A pilot experiment was conducted to determine the optimal temperature for evaluating heat tolerance in carrot. For this experiment, three widely grown commercial hybrids (coded as A, B, and C) and the cultivar Brasilia were selected to test seed germination under five temperatures (24.0, 32.5, 35.0, 37.5, and 40.0°C). 'Brasilia' was included because it is widely grown in the warmer climate of Brazil and is known to be relatively heat tolerant, whereas the three hybrids represent cultivars bred for more temperate production areas. The temperature at which statistically most significant differences were recorded between accessions, that being the lowest P value, was chosen as the optimal temperature to evaluate heat tolerance at the seed germination stage.
Germination assay. This experiment was conducted using a randomized complete block design with six replications and two treatments, that is, control (24°) and heat stress (35°). Twenty seeds from each carrot accession were placed on P5 filter paper in 60 · 15 mm petri dishes (Fisher Scientific, Waltham, MA). Petri dishes were added with 7 mL of distilled water and were placed in complete darkness at 24 ± 1°C for control in plastic bins and at 35 ± 1°C in an incubator (Fisher Scientific) for heat stress.
Evaluation under 35°C. Seed germination data were collected for a total of 10 d with measurements taken 2, 4, 6, 8, and 10 days after sowing. A seed was scored as germinated when the radicle had emerged and had a length of more than 1 mm. At each measurement time, any germinated seed was removed from the petri dish. Standard criteria for determining performance of carrot accessions under heat stress included final percent germination under nonstress conditions (PG Control ), final percent germination under heat stress (PG Heat ), AD, II, RHT, and HTI, as were used to evaluate salinity tolerance in carrot (Bolton and Simon, 2019). These measurements were calculated with the following equations: AD = PG Control -PG Heat ; II = 100 * (PG Control -PG Heat )/(PG Control ); RHT = PG Heat / PG Control ; HTI = (PG Heat *PG Control )/(PG Average ) 2 , whereas PG Average is the average percent germination of all carrot accessions evaluated under no heat stress.
Data analysis. Statistical mixed linear models (Eq. [1]) were used for analysis of variance (ANOVA) for six measurements related to seed germination on the basis of carrot accessions, origin of carrot accessions, and heat treatments.
where Y ij = the value of the measurements for the j th carrot accession in the i th replication where i = 1, ., 6, and j = 1, ., 289; m = total mean (constant), R i = effect of the i th replication (random effect) on the response measurement, A j = effect of the j th accession (fixed effect) on the response measurement, and Ɛ ij = effect of the experimental error associated with ij th observation. All analyses were performed in R. 3.4.4 (R Core Team, 2018). The lmer function in the lme4 package was used for ANOVA test (Bates et al., 2018). The mean separation analysis on the basis of region of origin of carrot accession was performed using least significant difference test function found in the agricolae package with alpha = 0.05 (De Mendiburu, 2014). Pearson rank correlations between measurements were calculated using the cor function found in the stats package (R Core Team, 2018).
The among-accession and within-accession variances were used for calculating broadsense heritability (H 2 ) as derived from Falconer and Mackay (1996) where s 2 G = genotypic (accessions) variance, s 2 P = phenotypic variance, s 2 E = variance due to experimental error, s 2 R = variance due to replication, and r = the number of replications for each treatment. Variance components were derived using formulas: s 2 G = (MSA -MSE) / r, s 2 E = MSE, s 2 R = (MSR -MSE) / n, with MSA = mean square accession, MSE = mean square error, MSR = mean square replication, r = number of replications, and n = number of accessions.

Results and Discussion
Optimal temperature for evaluation of heat tolerance at germination stage. Percent seed germination for the four cultivars evaluated in a preliminary study under nonstress conditions ranged from 44.0% to 84.0%, with a mean of 63.8%. Increasing temperature to 32.5°C reduced the mean percent germination to 57.2% and lowered the range from 45.0% to 80.0%. At 35°C, the mean percent germination was further reduced to 33.3% and lowered the range from 4.0% to 60.0%. At 37.5°C, only 'Brasilia' germinated, whereas at 40°C no germination occurred (Fig. 1). ANOVA for percent seed germination of the four carrot cultivars displayed a highly significant treatment effect (F = 154.63, P < 0.0001) and moderately significant accession effect (F = 4.78, P = 0.0044) on percent seed germination (Table 1). A significant difference between accessions was observed in the control temperature (24°C), whereas the most significant difference between accessions was recorded at 35°C (F = 13.18, P = 0.0002), indicating that 35°C is an optimal temperature to screen carrot germplasm for heat tolerance at the germination stage. At 37.5°C, only 'Brasilia' germinated, and at a low rate, whereas at 40°C there was no seed germination ( Table 2).
The results from this experiment indicate that a temperature of 35°C is an optimal temperature to test for heat tolerance, which was the same temperature used by Nascimento et al. (2008).
Germination assay. The average percent germination for the 293 carrot accessions under nonstress conditions ranged from 50.0% to 100.00%, with a mean of 81.6% and a standard deviation of 12.6%. When seeds were tested under 35°C, the average percent germination was reduced to 59.0% with a minimum value of 0.0%, a maximum of 97.5%, and a standard deviation of 19.5% (Fig. 2). These results indicate that heat stress significantly reduced percent seed germination in most carrot accessions. For all traits related to heat stress, there was a significant replication effect (P value < 0.0001) that may be attributed to variation within the incubator as reflected by the larger replication effect at 35°C than at 24°C (Table 3). Significant variation was recorded for the average percent germination among carrot accessions under control conditions (F = 8.35, P < 0.0001) (  Table 1). Percent germination also varied significantly under heat stress among carrot accessions (F = 4.88, P < 0.0001). PI 643114 (United States, 97.5%) had the highest percent germination value, whereas accessions PI 652208 (China), PI 652248 (Russia), PI 652400 (Turkey), and PI 652403 (Turkey) all had the second highest value (94.2%), indicating a high level of heat tolerance. Inbred B493B had the lowest percent germination value under heat stress (0.0%), followed by PI 652354 (Turkey, 4.2%), B7254B (7.5%), and PI 279764 (Syria, 10.8%), indicating that they were especially heat sensitive.
Several of the accessions with high percent germination under heat stress were cultivated PIs from diverse geographic regions. PI 643114 (United States), PI 652400 and PI 652403 (Turkey), PI 652208 (China), and PI 652403 (Russia) all had low AD and II values (-0.3% to 1.7%), high RHT values (0.95 to 1.0) and high HTI values (1.33 to 1.45). The most heat-sensitive carrot accessions were inbred lines and wild PIs. Inbred lines B493B and B7254B, and wild PIs from Syria (PI 279764) and Turkey (PI 652354) all had a percent germination less than 5.0% under heat stress, AD values from 50.8% to 86.7%, II values from 85.6% to 100.0%, and RHT and HTI values from 0.0 to 0.14. Surprisingly, the most tolerant accessions were not primarily from warmer regions of the world but were from very diverse geographic origins; and many tolerant accessions were cultivated varieties, suggesting that some degree of selection has occurred for heat tolerance in cultivated carrot in different parts of the world. Another interesting result was the low tolerance from several wild accessions native to regions with high levels of heat stress (Syria and Turkey), suggesting that adaptation to heat stress did not play a major role in carrot evolution and distribution based on these wild populations. In other species, sources of abiotic stress tolerance are often identified in crop wild relatives (Hajjar and Hodgkin, 2007). It appears that carrot differs from other species in this regard, as the least tolerant accessions are usually wild accessions whereas the most tolerant accessions are cultivated accessions. It should be noted that several wild carrot accessions were heat tolerant and several cultivated PIs were sensitive, contrary to general trends. That inbreds B493B and B7254B were highly sensitive was not surprising, as inbreeding depression may potentially make them more susceptible to the heat stress.
Interestingly, PI 652403 (Turkey) identified as heat tolerant in this study was also identified as one of the most salt tolerant accessions in a previous study, making this a potentially useful source of both salt and heat tolerance at the germination stage for carrot breeders (Bolton and Simon, 2019). Also, B493B had 0.0% germination under both salt stress in previous studies and heat stress in this study, indicating that this inbred line is very sensitive to two major forms of abiotic stress and can serve as a sensitive check in future studies.
AD in germination is a parameter used to measure heat stress (PG Control -PG Heat ). AD is a useful trait to identify accessions with very low tolerance to heat stress because it measures the actual reduction in germination. In this experiment, mean AD for all accessions evaluated was 22.6% and ranged from -13.0% to 86.7% ( Fig. 3A) with statistically significant variation observed among carrot accessions (F = 3.02, P < 0.0001) ( Table 3). PI 652154 (Netherlands, -13.0%) and PI 478370 (China, -12.3%) had the lowest AD values, indicating heat tolerance. A total of 15 accessions had negative AD values, indicating that heat stress increased percent germination compared with the nonstress conditions. Accessions displaying a negative AD value often had a low percent germination under control temperatures and low percent germination values under heat stress close to the overall group average (60%). These low AD value accessions might not be useful sources of heat tolerance for breeders, but do provide interesting material to investigate the genetic control of increased germination under heat stress. PI 652354 (Turkey, 86.7%) and PI 515992 (Hungary, 84.2%) had the highest AD values, indicating that heat stress greatly reduced germination and thus they were very heat sensitive.   Table 3) and had a range from 35.7% to 100.0% (Fig. 3B). PI 652154 (Netherlands, -35.7%) and PI 478370 (China, -22.3%) had the lowest II, agreeing with AD and indicating that heat stress increased germination for these accessions. B493B had a maximum II value of 100.0% followed by PI 652354 (Turkey, 95.6%), indicating that they are highly heatsensitive accessions.
RHT (RHT = PG Heat / PG Control ) is a useful criterion for evaluating heat stress, as it gives a way to account for the percent germination under the control. RHT was also significantly different among carrot accessions (F = 8.34, P < 0.0001) (Table 3) with a population mean of 0.74 and a range from 0 to 1.36 (Fig. 3C). PI 652154 (Netherlands, 1.36) and PI 478370 (China, 1.22) had the highest RHT values, whereas B493B, 7254B, PI 279764 (Libya), PI 515992 (Hungary), and PI 652354 (Turkey) all had low values (0.0 to 0.14). Similar to AD, accessions with high RHT values displayed low percent germination values under control conditions and thus provide a more useful resource for understanding the genetics of performing better under heat stress rather than useful breeding material.
Heat tolerance index [HTI = (PG Heat *PG Control )/(PG Average ) 2 ] is an important trait to consider, as it takes into account both the percent germination under heat stress and under control conditions while comparing each accession to the population average, thus giving a ranking among all accessions evaluated. An accession with a high HTI will have higher percent germination under both conditions, making it a useful accession to be selected for use in a commercial growing setting. HTI was significantly different among carrot accessions evaluation (F = 7.28, P < 0.0001) (Table 3) with a population mean of 0.74 and a range from 0.0 to 1.45 (Fig. 3D). PI 643114 (United States, 1.45) and PI 652208 (China, 1.4) had the highest HTI, suggesting that they were highly heat tolerant and had high percent germination under nonstress conditions, whereas inbred lines B493B and B7254B, along with PI 652354 (Turkey), had the lowest HTI values (0.0-0.06).
The accessions used in this study demonstrate a wide range of phenotypic variation for each of the heat stress parameters measured. Exposure to 35°C significantly reduced germination for most of the carrot diversity panel, agreeing with an earlier study (Nascimento et al., 2008). This evaluation reports a similar range of variation for heat tolerance as Nascimento et al. (2008), and evaluated nearly three times additional carrot accessions, including a broad range of cultivated open pollinated varieties. There were differences between these two evaluations for the four overlapping accessions ('Brasilia', PI 261782, PI 285613, and PI 537093) that may be attributed to differences in the seed lot and/or the number of replications that were used. These results suggest the value of evaluating a large number of diverse carrot accessions for heat stress, as has been suggested for salt stress in carrot (Bolton and Simon, 2019).
Heat tolerance according to geographic origin. Significant differences were observed for seed germination under control conditions and for all heat germination parameters among the 14 different geographic origins of carrot accessions (P values < 0.0001) ( Table 4). When comparing cultivated, wild, inbred, and hybrid accessions, accessions from Eastern and Central Asia demonstrated an average higher heat tolerance at the germination stage. Accessions from Eastern Asia had a mean AD of 13.96% and a range from -12.3% to 36.8%, a mean II of 14.5, and a range from -22.3% to 39.1%, a mean RHT of 0.86 and a range from 0.61 to 1.22, and a mean HTI of 0.82, and a range from 0.26 to 1.40. Accessions from Central Asia had similar values and ranges to those from Eastern Asia with a mean AD of 13.7% and a range from -5.0% to 30.5%, a mean II of 15.4% and a range from -17.8% to 44.1%, a mean RHT of 0.85 and a range from 0.56 to 1.17, and a mean HTI of 0.83 and a range from 0.29 to 1.38. Accessions from Eastern Africa and Southern Europe only included one and six accessions, respectively, but were among the most heat sensitive, having RHT and HTI values of 0.52 or lower along with AD and II values (41.4% to 50.6%) ( Table 5). Although accessions from Central and Eastern Asia were on average more heat tolerant, it is important to note the wide range of phenotypic variation within a single region and thus the need to evaluate multiple accessions from within a region when possible.
Heat tolerance according to domestication status and root color. No significant difference was observed for mean percent germination under nonstress conditions in comparing cultivated PIs (82.0%), wild PIs (80.2%), and inbred lines (78.4%) ( Table 6). Under heat stress, cultivated PIs demonstrated a significantly higher tolerance than the wild PIs and inbred lines, with a mean percent germination under heat of 64.3% and a range from 12.5% to 97.5%, a mean AD of 17.7%, mean II of 20.0%, mean RHT of 0.80, and mean HTI of 0.81. An important observation to note is that most wild PIs originated from Western Asia (Table 7) and the inbred lines are exclusively from the USDA breeding program, potentially accounting for the trends we observed. No significant difference in heat tolerance was observed between the wild PIs and inbred lines for all traits, and most accessions from  (Table 6). It is important to note that the number of samples for each color category were not equal and were not equally distributed across geographic origins, thus the trends reported here should be confirmed with larger sample sizes.
Broad-sense heritability. A high broadsense heritability (H 2 ) was observed for germination under nonstress conditions (H 2 = 0.88), for germination under heat stress (H 2 = 0.79), and for HTI (H 2 = 0.86), indicating a significant genetic basis for these traits. Heritability values for AD (H 2 = 0.66), II (H 2 = 0.66), and RHT (H 2 = 0.64) all were moderately high, suggesting a genetic basis for these traits. Interestingly, the heritability values for heat stress germination were lower than that observed in the same set of accessions for salt stress germination (Bolton and Simon, 2019). These differences in heritability suggest that either there is a smaller genetic component for heat tolerance germination in carrot, a larger environmental component that accounts for the phenotypic variation, or that it is a more genetically complex trait than salt tolerance germination with multiple genes responsible for the phenotype. These genetic factors warrant further investigation with the goal of identifying significant quantitative trait loci associated with heat tolerance at the germination stage.  Correlation among heat tolerance parameters, seed weight, and salt tolerance. Pearson correlation coefficients calculated for each of the heat tolerance parameters along with hundred seed weight (HSW) of each accession (Table 7) indicated that germination under nonstress conditions was highly correlated to the HTI (r = 0.74), but the correlation with germination under heat stress conditions (r = 0.50) was lower. Germination without stress was not strongly correlated to the other parameters evaluated (r = -0.20 to 0.16). These results indicate that percent germination under nonstress conditions does not predict heat tolerance germination in carrot. HSW had no correlation with percent germination under heat stress (r = 0.00) and a very weak negative correlation with germination under nonstress conditions (r = -0.20). These results were similar to those observed for salt tolerance germination where nonstress germination was also correlated with salt tolerance index (STI) (r = 0.54) and with stress salt stress (r = 0.40). Germination under salt stress was slightly  (Bolton and Simon, 2019). These data suggest that heat tolerance parameters and salt tolerance parameters follow similar trends, with heat tolerance having slightly stronger correlations among those parameters. Pearson correlation between HTI and the STI values calculated in Bolton and Simon (2019) display a strong correlation (0.69) between the two traits, suggesting that many of the accessions tolerant to one stress are also tolerant to the other. These results are not surprising, as heat stress and salt stress have similar effects on seed physiology and often cause similar stress responses. Although not evaluated in this study, it will be interesting to compare heat tolerance of these accessions in the field to heat tolerance at the seed germination stage and to determine if there is any correlation between the two studies.

Conclusions
This study identified a wide range of phenotypic variation for heat tolerance at the germination stage in a diverse collection of carrot germplasm. Five cultivated carrot accessions representing four countries (PI 643114, United States; PI 652208, China; PI 652248, Russia; and PIs 652400 and 654203, Turkey) were identified as the most heat-tolerant accessions, whereas inbred lines B493B and B7254B, along with wild accessions PI 279764 (Libya) and PI 652354 (Turkey) were identified as heat-sensitive accessions. The wide geographic range from which all the tolerant accessions originated was particularly surprising, as it suggests that heat tolerance has been under selection in multiple regions of the world where carrot is cultivated. This study adds to the current body of research for heat tolerance in carrot seed germination by identifying an optimal temperature for screening and measuring a high heritability of tolerance in a large collection of germplasm from diverse global regions. HTI was confirmed to be a particularly important measure of tolerance. This evaluation, coupled with recent evaluations of salt tolerance in the same diverse germplasm, provides valuable information for future studies of abiotic stress in carrot.  Table 1. Carrot accession, root color, country of origin, domestication status (DS), mean percent germination without heat stress (Nonstress) ± SE, mean percent germination with 35°C heat stress (Stress) ± SE, mean absolute decrease (AD), mean inhibition index (II), relative heat tolerance (RHT), mean heat tolerance index (HTI), hundred seed weight (HSW), and rank based on HTI. (Continued) Carrot accession, root color, country of origin, domestication status (DS), mean percent germination without heat stress (Nonstress) ± SE, mean percent germination with 35°C heat stress (Stress) ± SE, mean absolute decrease (AD), mean inhibition index (II), relative heat tolerance (RHT), mean heat tolerance index (HTI), hundred seed weight (HSW), and rank based on HTI.