Genetic diversity of winter wheat in Shaanxi province, China, and ...

Genetic Resources and Crop Evolution 49: 437–445, 2002.  2002 Kluwer Academic Publishers. Printed in the Netherlands.

437

Genetic diversity of winter wheat in Shaanxi province, China, and other common wheat germplasm pools Samuel P. Hazen 1,3 , Lieceng Zhu 2,4 , Hong-Sik Kim 1,5 , Guoshiun Tang 2 and Richard W. 1, Ward * 1

Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824, U.S. A.; Wheat Research Center, Shaanxi Academy of Agricultural Science, Yangling, Shaanxi P.R.C. 712100; 3 Current address: Plant Research Laboratory, MSU - DOE, East Lansing, MI 48824, U.S. A.; 4 Current address: Wheat and Barley Research Division, RDA National Crop Experiment Station, Suwon 441 -100, South Korea; 5 Current address: Plant Science Department, South Dakota State University, Brookings, SD 57007, U.S. A.; * Author for correspondence 2

Received 30 May 2001; accepted in revised form 12 December 2001

Key words: Chinese germplasm, Genetic diversity, Triticum aestivum, Wheat Abstract Cultivated Chinese wheat germplasm has been a valuable genetic resource in international plant breeding. Landraces endemic to China are a genetic resource that is distinct from other wheat germplasm. Patterns of genetic diversity among cultivated Chinese accessions and relationship to other germplasm pools are unknown, despite the proven value and potential novelty. The objective of this work was to determine the level of genetic diversity within improved Chinese germplasm in the context of several other wheat germplasm pools. We analyzed a set of improved accessions cultivated from the 1940s to the 1990s in Shaanxi province, China, using six simple sequence repeat (SSR) primer pairs and 30 restriction fragment length polymorphism - probe enzyme combinations (RFLP-PEC) previously used to characterize 21 geographically based germplasm pools. Shaanxi germplasm consists of three groups based on foreign introductions from Italy, Australia, Denmark, and Russia. There was a decrease in genetic diversity among Shaanxi accessions cultivated in the 1970s and 1980s to the 1990s, and accession classifications based on primary decade of cultivation were found to be significantly undifferentiated. The analysis of the mean genetic distance among 22 geographically based pools of germplasm suggests several regions are significantly undifferentiated. A vast majority of the total amount of variation was found within pools; therefore, pools appear to be largely differentiated based on small differences in band relative frequency and few if any unique bands. Previous studies have identified some Chinese landrace pools as morphologically and genetically unique. The Shaanxi pool does not have the same unique morphological or genetic features, nor is it more similar to the landrace pools than other improved germplasm pools. Abbreviations: AMOVA – Analysis of molecular variance, CHN-SW – Sichuan White wheat (China), CHN-TW – Tibetan Weedrace (China), CHN-XR – Xinjiang Rice wheat (China), CHN-YH – Yunnan Hulled wheat (China), CLINK – Complete linkage clustering methods, COP – Coefficient of parentage, EUS – Eastern US soft winter wheat, GD – Genetic distance, IWWSN – International winter wheat screening nursery, PCO – Principal coordinate analysis, PEC – Probe enzyme combination, PIC – Polymorphic information content, RFLP – Restriction fragment length polymorphism, SSR – Simple sequence repeat, UPGMA – Unweighted pair group method using arithmetic averaging, US-ER – Eastern U.S. soft red winter wheat, US-EW – Eastern U.S. soft white winter wheat, US-GP – U.S. Great Plains, US-W – Western U.S. soft white winter wheat, US-MSU – Michigan State University soft winter wheat.

438 Introduction Chinese germplasm has been shown to be both genetically and morphologically unique, as well as a valuable genetic resource in plant improvement. The evidence that describes Chinese germplasm as diverse is derived from the analysis of landraces (Ward et al. 1998; Yen et al. 1988; Kim and Ward 2000). Modern plant breeding in China began with pure line selection from local landraces, which descended from hexaploid wheat that entered the region as early as the middle of the third millennium BC (Feldman 1995). These diverse and unique landraces and subsequent selections were hybridized with foreign germplasm, mostly from Australia, Chile, Denmark, Italy, Korea, Romania, Russia, and USA (Yang and Smale 1996). Also, improvement of wheat in China has often emphasized introgression of alien species and local landraces into breeding material, a practice largely neglected outside of Asia (Rejesus et al. 1996). The early migration, foreign introduction, and alien introgression as well as molecular marker data, support the notion that cultivated germplasm in China may also be a unique and valuable set of germplasm. Utilized as a genetic resource in plant breeding, many valuable traits have been exploited. Genes for resistance to Fusarium head scab and barley yellow dwarf virus were recently discovered in Chinese germplasm (Van Ginkel et al. 1996; McGuire et al. 1995). The use of Chinese wheat germplasm is believed to have contributed novel yield genes to the International Center for the Improvement of Maize and Wheat (CIMMYT) germplasm, as well as genes for tolerance to heat and Karnal bunt, resistance to Septoria, lodging tolerance, fast grain fill, and immunity to stripe rust and bacterial resistance (Maarten Van Ginkel, personal communication); (Rajaram 1999). Despite the apparent value of Chinese wheat germplasm, little is known about the genetic relationship of accessions within Chinese germplasm and the relationship to other germplasm pools. Efforts have been made to characterize the genetic diversity in wheat using morphology (e.g., Sharma et al. (1998), Ritchie et al. (1987)), seed storage proteins (e.g., Cooke and Law (1998), Metakovsky and Branlard (1998)), coefficient of parentage (COP) (e.g., Cox et al. (1986), Nightingale (1996)), and DNA molecular markers (e.g., Bohn et al. (1999), Siedler et al. (1994)). Despite the large amount of information gathered concerning wheat genetic resources, these efforts do not necessarily have a cumulative effect.

These data are incongruous and relate specifically to germplasm within each particular study. For example, few inferences can be made between an analysis using COP and one using RFLP (restriction fragment length polymorphism). Data gathered using a different set of RFLP-PEC (probe-enzyme combination) or primer pairs within a PCR-based marker system are also not cumulative. A rigid and consistent use of a molecular marker system can overcome these problems by directly measuring DNA sequence variation without the need for parentage information. Kim and Ward (2000) conducted a survey of 292 winter wheat accessions from 21 germplasm pools. In the work presented here, the same RFLP-PEC used by Kim and Ward (2000) and six simple sequence repeat (SSR) flanking primer pairs were applied to the analysis of a set of accessions cultivated from the 1940s to the 1990s in Shaanxi province, China. These accessions were developed in Shaanxi province, where wheat is the most important crop, with an annual area of cultivation of nearly 1.7 million hectares (Yang and Smale 1996). The objective of this study was to quantify the genetic relationship of Chinese cultivated germplasm. This study was based on the analysis of the genetic relationship of cultivated wheat from a single region in China, which is temporally (1940s to the 1990s) and spatially (relative to 21 other germplasm pools) comprehensive. Materials and methods Plant material Twenty-four accessions of Triticum aestivum from Shaanxi Province, China were characterized for RFLP and SSR variability (Table 1). With the exception of one landrace (Maza mai), all accessions are inbred line cultivars and were developed in Shaanxi Province, China. Chinese Spring was included as a common genotype for comparison to other data sets. Abbondanza, an Italian cultivar, is a parent of Shaanxi cultivars and is considered here to be part of the Shaanxi germplasm. It was cultivated in the province in the 1980s. All of the Shaanxi accessions in Table 1 have winter or facultative growth habit. Molecular marker analysis Six SSR loci (flanked by primer pairs gwm 2, 6, 43, 155, 165, and 174) were used to genotype the 24

439 Table 1. Name, parentage, and decade of primary cultivation of the 24 T. aestivum accessions developed in Shaanxi, China. Name

Parentage

Decade of primary cultivation

Maza mai Bima 1 Bima 4 Xinong 6028 Shaannong 9 Fengchan 3 Aifeng 3 Xiaoyan 4 Xiaoyan 5 Shaanhe 6 Weimai 5 Weimai 4 Xiaoyan 6 Xiannong 151 Shaan 7859 Qinmai 6 Qinmai 9 Abbondanza Xiaoyan 168 Shaan 167 Shaan 229 Shaan 213 Xinong 65 Xinong 85

Landrace Maza mai / Quality Maza mai / Quality Jingyang 60 / Villa Glori Bima 5 / Xinong 6028 Danmai 1 / Xinong 6028 Xiannong 39 / 58(18)2 / / Fengchan 3 Fengchan 3 / Xiaoyan 759 ST 2422-464 / Xiaoyan 96 Bima 4 / Early Premium (Zaoyangmai) Kedong 51 / p6402 Nongda 311 / Fengchan 3 ST 2422-464 / Xiaoyan 96 Mianyang 75-20 / 75392-4-3-4 Predgornaja 2 (Shanqian mai) / /Ama /Abbondanza / 3 / Xibulai / / Fengchan 3 / 62(9)2-1 Zhengzhou 1 / Predgornaja 2 (Shanqian mai) Zhaomai 2 / 7410011-10 Autonomia / Fontarronco 1386 / Xiaoyan 6 Shaan 7587 / Tai 3429 Shaan 7853 / 80356 77-31 / Xiaoyan 6 Xiannong 39 / 58(18)2 / / Fengchan 3 Sumai 3 / Shaan 7859 / / 78(6)9-2 / 809(6)-5-6-1

1940s 1950s 1950s 1950s 1960s 1960s 1970s 1970s 1970s 1970s 1970s 1970s 1980s 1980s 1980s 1980s 1980s 1980s 1990s 1990s 1990s 1990s 1990s 1990s

¨ accessions (Roder et al. 1998). These primer pairs ¨ were associated with eight distinct loci (Roder et al. ¨ 1998). PCR was performed as described by Roder et al. (1998) and were separated by electrophoresis and stained as described in Hazen et al. (in press). The 30 probes used by Kim and Ward (2000) to screen 292 wheat accessions were used in combination with the HindIII restriction enzyme for RFLP analysis as described by Kim and Ward (1997). The RFLP data for the Shaanxi accessions were analyzed separately and jointly with corresponding data for the 292 accessions classified into 21 geography-based germplasm pools studied by Kim and Ward (2000). Statistical analysis The NTSYSpc version 2.02k software was used to generate genetic distance (GD) matrixes, create dendrograms and corresponding cophenetic matrixes, and calculate matrix correlations (Rohlf 1997). Genetic distance was calculated using Nei and Li (1979) computation: GD xy 512[2Nxy /sNx 1Nyd]; where N x and N y are the number of bands for each genotype, and N xy is the number of bands in

common between the two genotypes. Dendrograms were generated after cluster analysis with either the unweighted pair-group method using arithmetic averaging (UPGMA), the complete linkage (CLINK), or the flexible clustering procedures. Cophenetic matrixes were computed from the tree matrixes that generated the dendrograms. The matrix correlation (cophenetic correlation) between the original marker-based GD matrix and the corresponding cophenetic matrix was calculated to test the goodness of fit of a tree matrix and its associated dendrogram to the original distance matrix. Cophenetic matrix correlation values were interpreted as follows: ,0.7, very poor fit; 0.7 to 0.8 poor fit; 0.8 to 0.9, good fit; and 0.9 to 1.0, very good fit (Rohlf 1997). Principal coordinate analysis (PCO) was also carried out. The number and relative frequency of haplotypes were determined for each probe or SSR primer pair. Polymorphic information content (PIC) was calculated for each RFLP probe or SSR primer pair as described by Anderson et al. (1993). Cluster analysis of the mean pair-wise GD among accessions in different germplasm pools was used to analyze among-pool diversity patterns. The analysis of molecular variance (AMOVA) procedure of ARLEQUIN version 1.1 (Schneider et al. 1997) was used to estimate and test the significance of

440 within-and among-pool molecular covariances and FST . Tests of significance were based on ten thousand random permutations of the data for a given AMOVA. Results Molecular marker characteristics The results of Kim and Ward (2000) are critically compared to the RFLP-based genetic diversity of the Shaanxi province accessions and will be presented throughout the following sections. The thirty RFLP probes generated a combined total of 122 (53.7%) monomorphic and 105 (46.3%) polymorphic bands when all 24 accessions of the Shaanxi pool were considered. The relative frequency of monomorphic bands within each of the other 21 germplasm pools described in Kim and Ward (2000) ranged from 62 to 83%. All but one of the 227 bands reported here for the Shaanxi accessions were also found in one or more of the 292 accessions studied by Kim and Ward (2000). In comparison, the eastern United States soft red winter wheat pool (US-ER) had nine unique bands, but more than half of the other 21 germplasm pools had none (Kim and Ward 2000). Two bands were absent in the Shaanxi pool but were common or fixed in the other 21 germplasm pools. One of those bands (a 3.0-kb band produced by probe WG181) was also rare in the Chinese landrace pools considered in Kim and Ward (2000) and fixed or nearly fixed in all other pools. The second band that was uniquely absent from the Shaanxi pool (an 8.3-kb band produced by the probe WG 822) was virtually fixed in all other pools. Four bands were fixed in the Shaanxi germplasm and absent or rare in Kim and Ward (2000) other non-Chinese germplasm pools. Two of the those four bands were completely absent from non-Chinese pools but fixed in the Shaanxi pool and in the four Chinese landrace pools included in Kim and Ward (2000) study. Two other bands were fixed in the Shaanxi pool and absent or rare in all other pools. The only other accession that carried the BCD 1230 band was Ai lan mai, a tetraploid landrace from China. The average probe PIC value was 0.22 for the Shaanxi pool, whereas the range from other pools was 0.18 (Yunnan hulled wheat landrace from China (CHN-YH)) to 0.38 (Turkey landrace pool). Among the advanced germplasm pools, the range in average

probe PIC values was 0.19 (eastern US soft white winter wheat (US-EW)) to 0.34 (US-ER) (Kim and Ward 2000). Within the Shaanxi pool, fifteen (50%) of the probes generated a single haplotype (PIC 5 0.0) and one probe generated nine (PIC 5 0.843). The relative frequency of monomorphic probes in the other germplasm pools had a minimum of 0.13 for the Turkey pool, and a maximum of 0.63 for the CHIN-YH pool (Kim and Ward 2000). The six SSR primer pairs generated a total of 28 polymorphic and 2 monomorphic bands over all Shaanxi accessions. Primer pairs produced between one and five bands with a mean of 4.7. The SSR primer pairs generated between one and nine distinct haplotypes with a mean of 6.8. The PIC values for SSR primer pairs ranged from 0.0 to 0.84 with a mean of 0.62. Genetic diversity and structure of the Shaanxi germplasm pool Each of the Shaanxi accessions had a distinct haplotype relative to all of the 292 other accessions, when all 30 RFLP probes were considered jointly. Pairwise RFLP-based GD for the Shaanxi accessions varied from 0.004 (Bima1, Bima 4) to 0.068 (Qinmai 9, Shaan 167), with a mean of 0.041. The corresponding SSR-based GD values varied from 0.00 (Shaan 213, Xiaoyan 6) to 0.818 (Xiannong 151, Bima 4), with a mean of 0.443. The mean GD for the combined RFLP and SSR dataset was 0.076, with a range of 0.018 (Bima 1, Bima 4) to 0.115 (Weimai 5, Shaan 213). The existence of accession-based structure in the combined RFLP-SSR data for the Shaanxi accessions was investigated with one ordination (PCO) technique and three methods of hierarchical clustering (CLINK, flexible, and UPGMA). The separate cophenetic correlations for the CLINK, flexible, and UPGMA analyses were very poor, poor, and good at 0.70, 0.78, and 0.81, respectively. While the values vary, the clustering is in general agreement as seen by the correlation between the cophenetic matrixes from the UPGMA and CLINK and the UPGMA and flexible clustering were good and excellent at 0.83 and 0.94, respectively. All four analyses (PCO, UPGMA, flexible, and CLINK) suggested three major groups within the Shaanxi germplasm (figure not shown for either the PCO, flexible or CLINK analyses). The three postulated groups of related

441

Figure 1. Cluster analysis of 24 wheat accessions from Shaanxi Province, China, and accession Chinese Spring based on genetic distance (RFLP and SSR data). Brackets identify grouping of genetically similar accessions using a cluster cutoff of GD 5 0.065.

accessions are identified in the dendrogram derived from UPGMA cluster analysis (Figure 1). The large cluster (designated group 1 in Figure 1) consists of cultivars with a pedigree that includes an introduction from either Italy (st2422-464 and Villa Glori) or Australia (Quality). The older cultivars Bima 1, Bima 4, Shannong 9, Xinong 6028, Shaanhe 6, and the landrace Maza mai were subjectively divided into a subcluster (1a). Six of the nine accessions in the second subcluster (1b) have parents that are thought to carry a 1BL / 1RS wheat rye translocation. Of the other three accessions in subcluster 1b, two have uncertain parentage (Xiaoyan 168 and Xiaoyan 5), and one has a pedigree that gives no indication that it would carry 1BL / 1RS translocation (Xiaoyan 6). Cluster 2 is composed of cultivars derived from crosses that included Fengchan 3 (Danmai / / Jingyang 60 / Villa Glori) as a parent. Cluster 3 consists of three accessions (Abbondanza, Shaan 167, and Weimai 5) that have unique parents not found in other accessions. The classification system represented in Figure 1 was tested using AMOVA analysis of the RFLP-SSR data. Both among group covariance and the FST

parameter were significant, indicating that the deduced classification system reflects true structure among the Shaanxi accessions. Separate GD averages for accessions cultivated in the 1970s (n 5 6), 1980s (n 5 6), or 1990s (n 5 6) were calculated with the combined RLFP-SSR dataset. Diversity remained relatively constant in the first two decades (0.073 and 0.075, respectively) but decreased to 0.058 in the 1990s. Results of an AMOVA analysis of the decade of cultivation grouping revealed no evidence of among-decade differentiation (data not shown). Genetic diversity and structure of the combined Shaanxi and world germplasm The pool classification system used here and by Kim and Ward (2000) categorizes accessions according to institutional or geographic origin. AMOVA analysis of the RFLP data for all 316 accessions showed significant covariance within and among germplasm pools (Table 2). Although the majority (74%) of the total variation was within pools, the remaining variation (26%) supports the validity of this classifi-

442 Table 2. Analysis of molecular (RFLP) variance of the 22 wheat germplasm pool classifications based on geographic and breeding program origin. Source of variation

df

Sum of squares

Variance component

% Variation

Among germplasm pools Within germplasm pools Total

21 294 315

946.0 2200.6 3146.6

2.63*** 7.48*** 10.11

26.01 73.99

***Significant at P , 0.001

cation system. Differentiation of these accessions along the lines of the pool classification system is further supported by the results of AMOVA for all possible pairs of pools (Table 2). In only 20 of the total of 231 pair-wise pool comparisons was there insufficient evidence to conclude that two pools were different (a 5 0.001). The between-pool covariance and FST were both significant for all of the pair-wise AMOVA analyses involving the Shaanxi pool (Table 3). Pool-related structure was not, however, evident in the dendrogram (not shown) created from UPGMA cluster analysis of the complete pairwise GD matrix for all 316 accessions. The cophenetic correlation was 0.61, indicating that any structure in the GD

matrix was not evident in the dendrogram. There was a diffuse distribution across the dendrogram with few pools forming distinct clusters except the landrace groups. Accessions within a geographic pool generally cluster together, but membership in any one cluster was rarely exclusive. Accessions cluster primarily adjacent to members of the same pool as do twenty-two of the Shaanxi accessions. The French cultivar Montjoie, which has Australian parentage of Florence (a.k.a. Quality) and Aurore, was the only non-Shaanxi member of this cluster. The nearest cluster was comprised of eastern European germplasm ranging from Yugoslavia, Ukraine, Russia, and the US Great Plains (US-GP). The Shaanxi cluster was not associated with areas of the dendrogram

Table 3. The test of significance for the pairwise genetic diversity of germplasm pools. Values reflect the proportion of permutated pools leading to a F ST value larger or equal to true pairwise difference. Germplasm pool 1 (1) (2)

(3)

(4) (5) (6) (7)

(8)

(9) (10)

(11)

(12) (13) (14)

(15)

(16) (17) (18) (19) (20) (21)

(1) Shaanxi (2) Afghanistan (3) Argentina (4) France (5) Germany (6) Iran (7) IWWSN (8) ODESSA (9) CH-XR (10) Romania (11) Russia (12) CH-SW (13) CH-TE (14) Turkey (15) Ukraine (16) US-ER (17) US-EW (18) US-GP (19) US-MSU (20) US-W (21) Yugoslavia (22) CH-YH

0 0 0 0.009 0.009 0 0.009 0 0 0 0.009 0 0.009 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 0.089 0 0 0 0.297 0 0 0.128 0 0 0 0

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

0 0 0 0.782 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0.039 0 0 0 0

0 0 0 0 0 0

1

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

0 0 0 0.039 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 0 0 0

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

0.079 0 0.049 0.198 0 0 0 0.158 0 0 0 0 0 0 0

0.623 0 0 0 0.207 0 0 0.019 0 0 0 0

0 0 0 0 0 0 0 0 0

0 0 0 0.009 0 0 0 0

0 0 0 0 0

0 0 0 0

0 0 0

0 0

0

CHN-XR - Xinjiang Rice wheat (China); CHN-TW - Tibetan Weedrace (China); CHN-YH - Yunnan Hulled wheat (China); CHN-SW Sichuan White wheat (China); US-ER - Eastern U.S. soft red winter wheat; US-EW - Eastern U.S. soft white winter wheat; US-GP - U.S. Great Plains; US-MSU - Michigan State University soft winter wheat; US-W - Western U.S. soft white winter wheat; IWWSN - International Winter Wheat Screening Nursery

443 dominated by the Sichuan, Yunnan, Tibet, and Xinjiang Chinese landraces. The cultivar Weimai 5 clusters distantly from other Shaanxi accessions. The genetic distance among all 22 pools was analyzed by UPGMA cluster analysis of the mean genetic distance among pools. The cophenetic correlation for the resulting dendrogram was very high at 0.90. Four clusters were subjectively identified in the dendrogram, the largest of which (cluster 2) includes 15 pools divided into three subclusters. The first subcluster (2a) consists of Argentina, International Winter Wheat Screening Nursery (IWWSN), Odessa, Romania, Russia, Ukraine, US-GP, Shaanxi, Western US soft white winter wheat (US-W), and Yugoslavia. The second (2b) and third (2c) subclusters consist of eastern US and western European pools, respectively. The remaining pools are comprised of landrace material. The landraces from Turkey and the Xinjiang Rice wheat (CH-XR) cluster independently from all other pools. The three landrace groups, Sichuan white wheat (CH-SW), Tibetan weedrace (CH-TW), and CH-YH, make up the last cluster. Cluster and PCO suggest a subdivision in the data between primarily eastern European germplasm

(Romania, Russia, Ukraine, Odessa, US-GP, and Turkey) and primarily western European, South American and US (Germany, France, Argentina, USER, US-EW, US-W, Yugoslavia, and Shaanxi). The six remaining landrace pools cluster distantly from these two groups. Discussion The development of improved cultivars involves generating and evaluating new recombinant genotypes. Understanding crop genetic diversity aids in the calculated development and dissemination of improved cultivars. This benefit justifies the great effort expended to understand the population structure of domesticated plant species. However, analyses are often isolated where different methods are used to measure genetic distance among accessions. Investigations using COP and various biochemical and molecular marker systems or different markers within a system do not cumulatively add to the understanding of wheat genetic diversity as would be possible if evaluated using standardized methods. In

Figure 2. Cluster analysis of mean genetic diversity between 22 wheat germplasm pools classified by geographic or plant breeding program origin. Brackets subjectively identify natural grouping of genetically similar accessions using a cluster cutoff of GD 5 0.078 and subcluster designation at GD 5 0.070.

444 addition to comparing the genetic diversity among accessions within a pool, we should compare all pools to currently available and characterized accessions. The relative value of a germplasm pool in various breeding scenarios is in part a function of the diversity within the pool and its differentiation from other pools. In this study, we incorporated the Shaanxi germplasm into a previous analysis of 21 wheat germplasm pools using 30 RFLP-PEC. While RFLP are often well characterized and offer comparisons across species (Gale and Devos 1998; Van Deynze et al. 1995) they are somewhat laborious and costly and have lower expected heterozygosity or PIC values, lower effective multiplex ratios, and lower marker indices than SSR markers in several species (Bohn et al. 1999; Pejic et al. 1998; Powell et al. 1996). Fragments generated from SSR primers often are highly polymorphic, have well-characterized chromosome position, and have easily recognized allelic variants. The availability of automated sizing of fluorescent-labeled fragments and multiplexed gel electrophoresis (Diwan and Cregan 1997) present SSR as a prime candidate to characterize germplasm. Hexaploid wheat is believed to be founded on only a few polyploidization events (Talbert et al. 1998; Dvorak et al. 1998). Rapid dissemination of the species across Europe and Asia soon after domestication as early as the third and fourth millennium BC has led to an oligocentric distribution of genetic variation (Harlan 1975; Feldman 1995). The total amount of genetic diversity in common wheat appears to be spread geographically, and an unambiguous categorization of wheat genetic diversity measured using molecular markers has not been identified. The dendrogram of over 300 winter wheat accessions has diffuse and overlapping clusters of accessions based on geographic origin. The low level of polymorphism revealed by RFLP suggests an overall low level of genetic variation in wheat (Kim and Ward 2000). The analysis of the mean genetic distance among pools of germplasm suggests that many regions are significantly undifferentiated. Some germplasm pools such as hard red winter wheat pools in the former Soviet Union and the US-GP appear to be sub-samples of the same population. Most other pairwise comparisons of germplasm pools do not show a significant lack of differentiation. The genetic base of Shaanxi germplasm appears to be formed from Chinese landraces, primarily Maza

mai, landrace selection Jingyang 60, and a few important foreign introductions: Villa Glori, Quality, and Danmai 1. The cluster analysis indicates the importance of a few parents as described by Yang and Smale (1996). The early accessions are products of two-way and three-way crosses with Quality, Villa Glori, Early Premium, Maza mai, and Jinyang 60 a landrace selection. This material was then hybridized with Fengchan 3 or lines possessing the 1BL / 1RS wheat rye translocation. The most recently developed lines lack the historic introgression of foreign or unrelated plant introductions. This may be responsible for the large decrease in genetic diversity among Shaanxi accessions from the 1970s and 1980s to the 1990s. Accession classifications based on primary decade of cultivation were also found to be significantly undifferentiated. The absence of novel germplasm introduction into breeding material has resulted in the development of increasingly similar cultivars. This trend warrants action to reverse the apparent trend of reducing diversity among cultivars. A majority of the total amount of variation was found within pools. Pools appear to be differentiated based on small differences in band relative frequency and few if any unique bands. Kim and Ward (2000) suggest that the pool classification based on geographic area and plant breeding program reflects the true structure of wheat genetic diversity. The AMOVA verifies this hypothesis. However, not all pools are differentiated and the proportion of the total amount of variation responsible for those that are differentiated is relatively small. The Chinese landrace pools were collectively the most distinct and had several unique bands and banding patterns (Kim and Ward 2000; Ward et al. 1998). The evidence presented here suggests that improved Chinese germplasm does not have this same distinction from other germplasm pools. Shaanxi germplasm is typical of other gene pools described by Kim and Ward (2000) in amount and type of withinpool diversity. The Shaanxi pool is no more similar to the China landrace pools than to other improved pools.

Acknowledgements We thank Dilson Bisognin, Anne Plavonick-Jones, Mitch McGrath, Jim Hancock, Andy Jarosz, Dean Lehman, and Michael Retholtz for their assistance.

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