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Published online 19 March 2008
Published in Crop Sci 48:458-470 (2008)
© 2008 Crop Science Society of America
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Allelic Variation at the Vernalization Genes Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 in Chinese Wheat Cultivars and Their Association with Growth Habit

X. K. Zhanga,b, Y. G. Xiaoa, Y. Zhanga, X. C. Xiaa, J. Dubcovskyc and Z. H. Hea,d,*

a Institute of Crop Science, National Wheat Improvement Center/The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences (CAAS), 12 Zhongguancun South St., Beijing 100081, China
b College of Agronomy, Northwest Sci-Tech Univ. of Agriculture and Forestry, Yangling, Shaanxi 712100, China
c Dep. of Plant Sciences, Univ. of California, Davis, CA 95615, USA
d CIMMYT China Office, C/O CAAS, 12 Zhongguancun South St., Beijing 100081, China

* Corresponding author (zhhe{at}public3.bta.net.cn).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Information on the distribution of vernalization genes and their association with growth habit is crucial to understanding the adaptability of wheat (Triticum aestivum L.) cultivars to different environments. In this study, 278 Chinese wheat cultivars were characterized with molecular markers for the vernalization genes Vrn-A1, -B1, -D1, and -B3. Heading time was evaluated in a greenhouse under long days without vernalizaton. The dominant Vrn-D1 allele showed the highest frequency in the Chinese wheat cultivars (37.8%), followed by the dominant Vrn-A1, -B1, and -B3 alleles. Ninety-two winter cultivars carried recessive alleles of all four vernalization loci, whereas 172 spring genotypes contained at least one dominant Vrn allele. All cultivars released in the North China Plain Winter Wheat Zone were winter type. Winter (53.0%), spring (36.1%), and early-heading (10.9%) cultivars were grown in the Yellow and Huai River Valley Winter Zone. Most of the spring genotypes from this zone carried only the dominant Vrn-D1 allele, which was also predominant (64.1%) in the Middle and Lower Yangtze Valley Winter Zone and Southwestern Winter Wheat Zone. In three spring-sown wheat zones, all cultivars were early-heading spring types that frequently possessed the strongest dominant Vrn-A1a allele and combinations with other dominant Vrn gene(s). The Vrn-D1 allele is associated with the latest heading time, Vrn-A1 the earliest, and Vrn-B1 intermediate values. The information is important for breeding programs in countries interested in using Chinese wheats.

Abbreviations: PCR, polymerase chain reaction • UV, ultraviolet


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
COMMON WHEAT (Triticum aestivum L.) is one of the most widely cultivated food crops in the world and is grown over a wide range of elevations, climatic conditions, and soil fertility (Bushuk, 1998). The wide adaptability of wheat is largely governed by three groups of genetic factors— vernalization (Vrn) genes (vernalization requirement), photoperiod (Ppd) genes (photoperiod sensitivity), and genes controlling earliness per se (Eps) (Kato and Yamagata, 1988)—that act together to determine flowering time and hence the basic adaptation of a genotype for a particular environmental condition (Worland, 1996; Worland et al., 1998). Vernalization genes determine growth habits, which divide wheat into winter and spring classes. Winter cultivars are mainly adapted to areas with average January temperature between –7 and 4°C, whereas spring cultivars are adapted to areas with temperatures below or above this range (Iwaki et al., 2000, 2001). The different frequencies of Vrn alleles observed in different parts of the world suggest that these allele combinations have an adaptive value (Gotoh, 1979; Stelmakh, 1990; Iwaki et al., 2000, 2001; Goncharov, 1998; Stelmakh, 1998). For example, the dominant Vrn-A1 allele is frequently observed in improved cultivars from Europe and Siberia, whereas higher frequencies of dominant Vrn-D1 allele were found in commercial cultivars from countries situated nearer the equator (Stelmakh, 1990), such as Japan (Gotoh, 1979), the Central Asian Republic of the Former Soviet Union (Stelmakh, 1990), and China (Iwaki et al., 2000, 2001), or in Mediterranean climates (Fu et al., 2005). Important germplasm from the International Maize and Wheat Improvement Center (CIMMYT) have also been classified for their phasic gene constitution, allowing conclusions about the frequencies of occurrence of certain gene combinations (Van Beem et al., 2005). Therefore, an understanding of the vernalization genes present in wheat breeding programs is useful when developing cultivars broadly adapted to different regions.

Various studies showed that the vernalization requirement is genetically controlled by at least five loci, Vrn-A1 (formerly Vrn1), Vrn-B1 (Vrn2), Vrn-D1 (Vrn3), Vrn4, and Vrn-B3 (Vrn5), in global sets of commercial cultivars (Pugsley, 1971, 1972; McIntosh et al., 1998; Goncharov, 2003; Yan et al., 2006). The three major vernalization genes Vrn-A1, Vrn-B1, and Vrn-D1 are located on the homeologous chromosomes 5A, 5B, and 5D in common wheat, respectively (Pugsley, 1971; Law et al., 1976; Worland, 1996; Barrett et al., 2002; Yan et al., 2003), and Vrn-B3 is located on chromosome arm 7BS (Law and Wolfe, 1966; Yan et al., 2006). The spring alleles from these genes are epistatic to the winter alleles, and, therefore, the winter habit is observed only when all the genes have recessive alleles (Pugsley, 1971). The Vrn-A1a allele is the most potent allele for spring growth habit, providing complete insensitivity to vernalization, whereas Vrn-B1, Vrn-D1, and Vrn4 result in a partial elimination of the vernalization requirement (Pugsley, 1971, 1972).

Detection of vernalization genes by traditional genetic methods is time consuming. Fortunately, the recent cloning of wheat vernalization genes (Yan et al., 2003, 2006) has facilitated the development of gene-specific markers or functional markers (also known as perfect or diagnostic markers). These markers provide a unique opportunity to screen large collections of wheat germplasm for allelic diversity at the Vrn genes. Yan et al. (2003) used diploid wheat T. monococcum (2n = 14, AmAm) to clone the Vrn-Am1 gene, using a positional cloning approach to show that this gene is similar to the Arabidopsis meristem identity gene APETALA1. The different dominant Vrn-A1 alleles were identified in polyploid wheats. The most abundant one in common wheat, Vrn-A1a, has an insertion of a foldback repetitive element and a duplicated region in the promoter (Yan et al., 2004). The less frequent Vrn-A1b allele shows several single nucleotide polymorphisms and deletions in the promoter region (Yan et al., 2004), whereas the rare Vrn-A1c allele has a large deletion in the first intron (Fu et al., 2005). The Vrn-A1c allele was found only in the spring hexaploid landrace IL369 from Afghanistan but is common among tetraploid spring genotypes. The dominant Vrn-B1 and Vrn-D1 alleles for spring growth habit are also characterized by large deletions in the first intron of the same gene (Fu et al., 2005). A dominant allele for spring growth habit was recently identified in the cultivar Hope and was designated Vrn-B3 on the basis of its orthology with the barley Vrn-H3 gene (Yan et al., 2006). The dominant Vrn-B3 allele has a retrotransposon insertion in the promoter region of a gene similar to Arabidopsis FT (Yan et al., 2006).

China is the largest wheat producer in the world. Ten major wheat agroecological zones have been recognized in China (Fig. 1 ) on the basis of differences in wheat types, growing season, presence of major biotic and abiotic stresses, and cultivar responses to temperature and photoperiod (Zhuang, 2003). At present, autumn-sown wheats account for about 90% of production and acreage and include zones I (4%), II (60%), III (13%), IV (10%), and V (minor area of production). Spring-sown wheats represent 7% of the wheat acreage in China and are grown in zones VI, VII, and VIII. Zones IX and X cover less than 3% of the total wheat area and include both spring- and fall-sown wheats. Average January temperatures and wheat-growing periods vary greatly among different Chinese wheat regions (Jin, 1986, 1997; Zhuang, 2003). The spring-sown wheat regions are well defined, and only spring cultivars are found in these regions. However, the autumn-sown regions include both winter and spring cultivars, resulting in some confusion in cultivar classification. As an example, spring-type cultivars from Zones II and III are sometimes planted in the northern part of Zone II, resulting in severe losses due to winter damage (Jin, 1986, 1997; Dong and Zheng, 2000; Zhuang, 2003). Therefore, information on the distribution of vernalization genes in Chinese wheats is crucially important for developing widely adapted cultivars and for providing information to extension workers and farmers.


Figure 1
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  		Figure 1. Distribution of growth habit and vernalization allele combinations among China's different wheat (Triticum aestivum L.) zones.

 
Previously, vernalization genotypes and growth habits of 42 Chinese wheat landraces were studied by crossing them with near-isogenic lines of ‘Triple Dirk’ carrying different vernalization genes (Gotoh, 1979; Iwaki et al., 2000, 2001). However, the vernalization genotypes and growth habits of wheat cultivars released in China from the 1960s to the present are mostly unknown. The aims of this study are (i) to characterize allelic variations at the four vernalization loci Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 among the major Chinese wheat cultivars released since the 1960s using molecular markers; (ii) to test heading times of these cultivars under controlled conditions; and (iii) to analyze the relationships between vernalization genotypes, geographic distribution, and planting times for these cultivars. This information is expected to be useful for improving the adaptation of wheat cultivars in Chinese breeding programs targeting different environments and for foreign breeding programs interested in using Chinese wheat germplasm.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material
A total of 278 Chinese wheat cultivars collected from eight major zones were used to identify their vernalization genotypes using polymerase chain reactions (PCR) methods, and the growth habits of 266 of them were assessed in the greenhouse (see Table A1 in Appendix). They include landmark landraces, leading cultivars, and 12 well-known introductions that had significant impact on Chinese wheat production and breeding after 1960. The number of entries in various zones was based on wheat acreage and number of cultivars developed by the local breeding programs (Table 1 ). Cultivars Thatcher (Vrn-A1a), Chinese Spring (Vrn-D1), and Hope (Vrn-B3) were used as controls.


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  		Table A1. Growth habit and allelic variation at the Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 loci in Chinese and certain introduced wheat (Triticum aestivum L.) cultivars.

 

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  		Table 1. Distribution of growth habits and combination of dominant alleles at Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 loci in various Chinese wheat (Triticum aestivum L.) zones.

 
DNA Extraction and Molecular Marker Analysis
Genomic DNA was extracted from seeds following the procedure of Gale et al. (2001). Sequences of nine specific primer sets for the amplification of allelic variations at Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 loci have been published before (Yan et al., 2004, 2006; Fu et al., 2005) and are summarized in Table A2 in the Appendix. These primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China; http://www.sangon.com).


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  		Table A2. Primer sequences, expected polymerase chain reaction (PCR) band sizes, and PCR conditions for detecting alleles at the Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 loci.

 
Polymerase chain reaction was performed in an MJ Research PTC-200 thermal cycler (Waltham, MA). The PCR conditions for the primer pair VRN1AF and VRN1-INT1R were as follows: 1X PCR buffer with 1.5 mM of MgCl2, 150 µM of each dNTPs, 2 pmol of each primer, 1 unit of Taq DNA polymerase (Tiangen Biotech Co., Beijing, China) and 50 to 100 ng of template DNA in 20 µL of final volume. Thermocycling conditions were an initial denaturation at 94°C for 10 min, followed by 38 cycles of 45 s at 94°C, 45 s at 50°C, 1 min at 72°C, and with a final extension step at 72°C for 5 min. Amplified PCR fragments were separated on a 2.5% agarose gel at 80 V for 4 to 5 h, stained with ethidium bromide, and visualized using ultraviolet (UV) light. Thatcher (Vrn-A1a) and Chinese Spring (vrn-A1) were used as controls in the test. For the other eight primer sets we used 10 pmol of each primer and the PCR conditions were similar to those for the VRN1AF/VRN1-INT1R primer pair except for the annealing temperatures and extension times (see Table A2). The amplified PCR fragments were separated on a 1% agarose gel at 150 V, stained with ethidium bromide, and visualized using UV light.

Greenhouse Experiment
Heading times of 266 wheat cultivars were evaluated following the methods of Stelmakh (1987), Iwaki et al. (2001), and Beales et al. (2005) with minor modifications. Cultivars were grown in a greenhouse under a 16-h-daylength regime and a temperature of 18 ± 3°C to avoid natural vernalization. For each cultivar, five germinated seeds were sown in soil-filled containers at a space of 2.5 cm between plants in a row and 6 cm between rows. Days to heading were recorded during 6 mo in the greenhouse.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Allelic Frequencies at the Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 Loci
The specific allele combinations identified in the 278 wheat cultivars are shown in Table A1. First, all cultivars were tested with primers VRN1AF and VRN1-INT1R for the Vrn-A1 promoter region. A total of 68 cultivars from Zones IV, VI, VII, VIII, and X showed PCR fragments identical to those in Thatcher (965 bp and 876 bp), indicating the presence of the dominant Vrn-A1a allele (Fig. 2A , Table A1). Only eight cultivars (Fan 7, Chuanyu 12, and Jingmai 11 from Zone IV, Dabaipi from Zone VII, Longchun 8, Longchun 21, and Ganmai 8 from Zone VIII, and Xinchun 9 from Zone X) showed the 714-bp fragment characteristic of the Vrn-A1b allele (Fig. 2A, Table A1). The remaining 202 cultivars exhibited the 734-bp fragment characteristic of the dominant allele Vrn-A1c or the recessive vrn-A1 allele (Fig. 2A). To distinguish between these two alleles, all cultivars were tested using the two primer pairs Intr1/A/F2 and Intr1/A/R3, and Intr1/C/F and Intr1/AB/R, for the Vrn-A1 first intron. A 1068-bp fragment was amplified in all cultivars tested using the primer pair Intr1/C/F and Intr1/AB/R (Fig. 2C), whereas no PCR product was produced using primer pair Intr1/A/F2 and Intr1/A/R3 (Fig. 2B). These results indicate that the large intron 1 deletion (Vrn-A1c allele) was not present in the Chinese cultivars and that the 202 cultivars with the 734-bp amplification product carried the recessive vrn-A1 allele (Table A1).


Figure 2
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  		Figure 2. Polymerase chain reaction amplification using primer pairs (A) VRN1AF and VRN1-INT1R, (B) Intr1/A/F2 and Intr1/A/R3, and (C) Intr1/C/F and Intr1/AB/R to detect alleles at the Vrn-A1 locus. 1, Chinese Spring (vrn-A1); 2, Jing 411 (vrn-A1); 3, Yumai 2 (vrn-A1); 4, Thatcher (Vrn-A1a); 5, Xinkehan 9 (Vrn-A1a); 6, Longchun 21 (Vrn-A1b); 7, Gan 630 (Vrn-A1a); 8, Shaan 229 (vrn-A1).

 
A total of 73 cultivars have the dominant Vrn-B1 allele as indicated by the amplification of a 709-bp fragment using primers Intr1/B/F and Intr1/B/R3 (Fig. 3A ). The other 205 cultivars showed a 1149-bp amplification product with primers Intr1/B/F and Intr1/B/R4, which is characteristic of the recessive vrn-B1 allele (Fig. 3B and Table A1).


Figure 3
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  		Figure 3. Polymerase chain reaction amplification using primer pairs (A) Intr1/B/F and Intr1/B/R3 and (B) Intr1/B/F and Intr1/B/R4 to detect the dominant (Vrn-B1) and recessive (vrn-B1) alleles at the Vrn-B1 locus, respectively. 1, Abbondanza; 2, Dongfanghong 3; 3, Mentana; 4, Mazhamai; 5, Zhengmai 9023; 6, Mianyang 11; 7, Mianyang 15; 8, Miannong 4; 9, Kefeng 3; 10, Xinkehan 9; 11, Longmai 20; 12, CI12203; 13, Jinchun 14; 14, Xuzhou 25; 15, Chinese Spring; 16, Xinchun 12.

 
A 1671-bp fragment was generated from 105 cultivars using primer pair Intr1/D/F and Intr1/D/R3 (Fig. 4A ), demonstrating that they carried the dominant Vrn-D1 allele. Amplification of DNA from the other cultivars using primers Intr1/D/F and Intr1/D/R4 showed a 997-bp band characteristic of the recessive vrn-D1 allele (Fig. 4B and Table A1).


Figure 4
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  		Figure 4. Polymerase chain reaction (PCR) amplification using primer pairs (A) Intr1/D/F and Intr1/D/R3 and (B) Intr1/D/F and Intr1/D/R4 to detect the dominant (Vrn-D1) and recessive (vrn-D1) alleles at the Vrn-D1 locus, respectively. 1, Chinese Spring; 2, Beijing 10; 3, Jing 411; 4, Bima 4; 5, Abbondanza; 6, Neixiang 36; 7, Jinan 2; 8, Zhoumai 18; 9, Shijiazhuang 8; 10, Shaannong 7859; 11, Yumai 2; 12, Lumai 1; 13, Lumai 14; 14, Zhengmai 9023; 15, Mentana; 16, Emai 6.

 
The dominant Vrn-B3 allele, defined by the amplification of a 1.14-kb fragment with primers VRN4-B-INS-F and VRN4-B-INS-R, was found only in cultivars Longfumai 1 and Liaochun 10 from Zone VI (Fig. 5A ). All other cultivars showed a 1.2-kb amplification fragment using primers VRN4-B-NOINS-F and VRN4-B-NOINS-R, which is characteristic of the recessive vrn-B3 allele (Fig. 5B, Table A1).


Figure 5
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  		Figure 5. Polymerase chain reaction amplification using primer pairs (A) VRN4-B-INS-F and VRN4-B-INS-R and (B) VRN4-B-NOINS-F and VRN4-B-NOINS-R to detect dominant (Vrn-B3) and recessive (vrn-B3) alleles at the Vrn-B3 locus, respectively. 1, Chinese Spring; 2, Shijiazhuang 407; 3, Lumai 21; 4, Xi'an 8; 5, Jinan 2; 6, Shaanong 7859; 7, Sumai 3; 8, Neixiang 36; 9, Neimai 19; 10, Gan 630; 11, Liaochun 10.

 
Geographic Distribution of the Different Allele Combinations
The frequencies of the different Vrn allele combinations varied greatly across different wheat agroecological zones (Table 1, Fig. 1). Cultivars with recessive alleles at all the analyzed Vrn loci represent 38.1% of the cultivars and are mainly concentrated in Zones I, II, and X (Table 1). The other 61.9% includes cultivars with at least one dominant Vrn allele, which can be classified as spring. These cultivars are found mainly in Zones III, IV, VI, VII, VIII, and X (Table 1).

Among the cultivars with at least one dominant Vrn allele, the frequencies of the different alleles varied across regions (Table 1). The dominant Vrn-B3 allele is present only in two cultivars from zone VI. Among the Vrn-1, alleles Vrn-D1 showed the highest frequency, followed closely by dominant Vrn-A1 and Vrn-B1 alleles (Table 1). The dominant Vrn-A1 allele is not presented in Zones I, II, and III, and its frequency is low in Zone IV. However, high frequencies are observed in Zones VI, VII, VIII, and X (Table 1, Fig. 1). The dominant allele Vrn-B1 is not present in Zone I, and low frequencies are observed in Zones II and III. However, high frequencies are observed in Zones IV, VI, VII, VIII, and X. The dominant allele Vrn-D1 is not present in Zone I, but is present at relatively high frequencies in Zones II, III, IV, VI, VII, VIII, and X.

Among the four autumn-sown wheat zones (I, II, III, and IV), the frequency of dominant Vrn-D1 allele is the highest, followed by Vrn-B1 and Vrn-A1 (Vrn-B3 is absent) (Fig. 6 ). In contrast, in three spring-sown wheat zones (VI, VII, and VIII) the frequency of the dominant Vrn-A1 allele is the highest, followed by Vrn-B1 and Vrn-D1, respectively (Fig. 6). Vrn-B3 frequency (2.9%) is the lowest.


Figure 6
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  		Figure 6. Percentage of different vernalization allele combinations among spring-sown spring and autumn-sown spring cultivars in China.

 
Frequencies of the different combinations of vernalization genes were also very different among the various wheat agroecological zones (Table A1 and Table 1). In brief, nine combinations of dominant Vrn alleles were identified. Among them, the Vrn-D1 allele alone was the most frequent (72 cultivars), followed by the Vrn-A1Vrn-B1 (36 cultivars) combination. The distribution of the different allele combinations across the different zones is described in Table 1. In summary, most cultivars released in the autumn-sown wheat regions of south China (Zones III and IV) and north China (Zone II) possessed Vrn-D1 as a single dominant allele. In contrast, in spring-sown wheat regions, cultivars carried the strongest dominant Vrn-A1 alleles, and the majority of them included additional dominant Vrn alleles at the Vrn-B1, Vrn-D1, and Vrn-B3 loci. On the basis of the vernalization alleles found in this study, the vernalization requirement can be ranked from strongest to weaker from Zone I, Zone II, Zone III, Zone IV, with the weakest requirement in the spring-sown spring wheat regions (Zones VI, VII, and VIII).

Growth Habit
Heading dates showed a continuous distribution from 30 d to more than 6 mo after planting in the greenhouse (Table A1). Of 266 cultivars tested in the greenhouse, the 92 cultivars that failed to head within 109 d all possessed recessive vernalization alleles at the four Vrn loci as identified by the PCR markers. Most of them were classified as winter cultivars in the literature (Jin, 1986, 1997; Zhuang, 2003). Among the 174 cultivars that headed within 109 d (early heading), 164 carried at least one of the tested dominant vernalization alleles and were classified as spring. The other cultivars, nine from Zone II (Jimai 36, Taishan 1, Lumai 23, Laizhou 953, Weimai 8, Xinmai 9408, Yumai 66, Yumai 70, Xuzhou 14) and one from Zone III (Emai 6), carried recessive alleles at the four vernalization loci. The most likely explanation for this discrepancy is the presence of an unknown allele at the four loci characterized in this study or the presence of a spring allele at the Vrn4 locus not included in this survey because the gene is still unknown.

All 32 cultivars released in Zone I headed after 109 d, had all three recessive vrn-1 alleles, and were classified as winter. In Zone II, of 75 cultivars tested in the greenhouse, 44 (58.7%) headed after 109 d. Although both winter and spring types are found in all provinces of Zone II, the late-heading cultivars were mainly cultivated in the provinces of Shandong (79.2%), Shaanxi (63.6%), and Anhui (100.0%), whereas the early-heading cultivars were mostly present in the provinces of Henan and Jiangsu. Most of the cultivars from Zones III and IV ( >94%) headed before 109 d. The frequency of early-heading genotypes increased gradually from north to south in the autumn-sown regions. In Zones VI, VII, and VIII, all 68 cultivars tested in the greenhouse headed within 109 d. In Zone X, the frequency of early-heading genotypes was 51.9%. Therefore, it was concluded that spring cultivars in China are more frequent in the high-latitude regions (spring sowing) and in the low-latitude area with warm winters (autumn sowing). Winter cultivars are frequently present in the middle-latitude area with relatively cold winters (autumn sowing).

Relationships between Vrn Allele Combinations and Growth Habits
The relationships between vernalization genotypes and heading times in the absence of vernalization are shown in Table 1. The 92 late-heading (winter) cultivars all carried recessive alleles at the four vernalization loci. Different combinations and proportions of vernalization alleles were found in the other 172 early-heading (spring) cultivars. Single dominant alleles were observed for the Vrn-A1 (11.0%), Vrn-B1 (6.4%), or Vrn-D1 (41.9%). We also observed two gene combinations, including Vrn-A1–Vrn-B1 (20.9%), Vrn-A1–Vrn-D1 (4.6%), and Vrn-B1–Vrn-D1 (7.6%), and three dominant allele combinations, including Vrn-A1Vrn-B1Vrn-D1 (6.4%) and Vrn-A1Vrn-B1Vrn-B3 (0.6%). In addition, one very early heading cultivar (Liaochun 10) carried all four dominant alleles (Vrn-A1Vrn-B1Vrn-D1Vrn-B3).

Days to heading among the different combinations of dominant vernalization alleles are described in Table 1. In summary, the earliest cultivars were those carrying three to four dominant alleles, including the rare Vrn-B3 allele (average 30 to 31 d to heading), followed by the one-, two- or three-gene combinations, including Vrn-A1 but not Vrn-B3 (average 38 d to heading). Lines carrying the Vrn-B1/Vrn-D1 allelle combination headed approximately 42 d after sowing, whereas those carrying only the Vrn-B1 (average 47 d) or Vrn-D1 (average 54 d) were among the latest spring cultivars. On the basis of these data, the strength of the dominant spring Vrn-1 alleles can be ranked as Vrn-A1 > Vrn-B1 > Vrn-D1. Vrn-B3 resulted in the earliest heading times in combination with other dominant Vrn1 alleles.


   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Effectiveness of Molecular Markers for Identifying Vernalization Alleles
Functional markers (also known as perfect markers) are derived from polymorphic sites within genes that directly affect phenotypic trait variation, and they are ideal tools for marker-assisted selection (Bagge et al., 2007). The vernalization gene markers developed by Yan et al. (2004, 2006) and Fu et al. (2005) are likely functional markers. The observed heading times in the greenhouse experiment and the growth habit determinations from the literature (Jin, 1986, 1997; Dong and Zheng, 2000; Zhuang, 2003) were consistent with the Vrn genotypes. However, 10 of the 174 cultivars showed inconsistent results, with early heading in the greenhouse but recessive alleles present at the four Vrn alleles characterized in this study. These exceptions are most likely due to the presence of the Vrn4 locus, which is known to be present in Chinese landraces (Iwaki et al., 2000, 2001). Unfortunately, this gene has not been cloned, and no markers are currently available to screen these cultivars. An alternative possibility is the presence of new mutations at the Vrn loci included in this study outside the region tested with the available primers or the presence of alleles for spring growth habit at unknown vernalization genes. Further investigation of these exceptions may provide new insights on wheat vernalization genes.

Additional exceptions were the early-heading Chinese landraces Jiounong 2 and Ganmai 11 from Zone VIII, which showed three amplification products with primers VRN1A and VRN1-INT1R (data not shown). This result suggests that these landraces may carry a new Vrn-A1 allele. However, further investigation is needed to confirm this assumption.

In spite of these few exceptions, the distribution of dominant Vrn-A1, Vrn-B1, and Vrn-D1 alleles based on molecular markers (Yan et al., 2004; Fu et al., 2005) among modern Chinese cultivars is similar to that reported before for a smaller set of Chinese landraces using crosses with near-isogenic lines of ‘Triple Dirk’ (Gotoh, 1979; Iwaki et al., 2000, 2001). These results indicate that these molecular markers can be effectively used to detect allelic variations at these four vernalization gene loci.

Reasons for Different Distributions of Growth Habit and Vernalization Genotypes among Wheat Zones of China
The average January temperatures increase from Zone I to II, from II to III, and from III to IV, whereas the length of growth periods decreases in the same direction (Dong and Zheng, 2000; Zhuang, 2003). The frequency of dominant vernalization alleles gradually increases in the same direction (Fig. 1). Only winter cultivars with recessive alleles at the four vernalization loci can survive in the cold winters of Zone I, whereas almost all cultivars released in Zones III and IV with warmer winters are spring types. Most of them carry the single dominant Vrn-D1 allele, due to the wide utilization of some breeding parents, including Mentana with Vrn-D1 in Zones III and IV (Stelmakh, 1990; Zhuang, 2003). The Vrn-D1 allele is the weakest of the dominant Vrn-1 alleles and has a residual requirement for vernalization that is well suited for fall planted wheats in regions with mild winters.

Zone II is located in the middle of Zones I, III, and IV (Fig. 1). The frequency of winter cultivars planted in Zone II is lower than that in Zone I and higher than those in Zones III and IV. In general, cultivars from the northern part of Zone II have better winter hardiness or freezing resistance than cultivars from the southern part of Zone II (Zhuang, 2003). Winter wheats cannot be cultivated in Zones VI, VII, and VIII, where the average minimum temperature in January and February is too low. In these regions spring wheats are planted in spring to avoid the colder conditions (Wilsie, 1962). The length of growth periods in spring-sown regions (VI, VII, and VIII) is shorter than that in autumn-sown regions (Dong and Zheng, 2000; He et al., 2001; Zhuang, 2003). These characteristics may explain the high frequency of cultivars with the dominant Vrn-A1 allele in these spring-sown regions, because this allele has the strongest insensitivity to vernalization, conferring a very early heading time that is essential for the adaptation to short growing seasons. Heading time can be further accelerated by the presence of multiple Vrn alleles. Lines with multiple Vrn alleles are frequent in Zone VI, which has the shortest growing season in China. This region is also the only one where the very early heading Vrn-B3 allele from Hope was found. Interestingly, Hope is not presented in the pedigrees of Liaochun 10 and Longfumai 1 (Zhuang, 2003). In Zone X, the average January temperatures are very low, so spring cultivars are planted in spring and winter cultivars in autumn. Winter wheats can survive largely because of snowfall in this region.

The large differences in the frequencies of dominant vernalization alleles observed across the different agroecological regions suggest that these distributions are largely determined by environmental factors. Particularly important are the differences in average January temperatures and the length of growth periods among different zones. Cultivars with the most suitable vernalization genes are maintained through long-term natural selection and breeder selection in the wheat breeding programs. In addition to vernalization genes, other genes such as the photoperiod genes and the earliness per se genes play important roles in the determination of heading time in different cultivars. They are the reasons why distributions of growth habit and vernalization genotypes between Chinese and other countries' wheats are different (Stelmakh, 1998; Iwaki et al., 2000, 2001; Fu et al., 2005). The effect of dominant spring Vrn-1 alleles on heading time in this study differs from that described by Stelmakh (1993). Stelmakh (1993) used three genetic backgrounds to study effects of Vrn-1 genes on heading date in the field, showing that the Vrn-B1 allele was associated with the latest heading time, Vrn-A1 with the earliest, and Vrn-D1 with intermediate values. The difference could be due to dissimilar genetic backgrounds of cultivars and environments. Therefore, further investigation is needed to understand the distribution of these associated genes and their interactions with the vernalization genes in determining the adaptability of wheat cultivars to the different agroecological regions.

Classification of Autumn-Sown Wheat Zones in China
A genetic definition of growth habit (winter vs. spring) of wheat cultivars on the basis of vernalization genotype is superior to the customary definition based on sowing time (Crofts, 1989). Autumn-sown wheats in China are traditionally referred to as winter types (Jin, 1986, 1997; Zhuang, 2003), and, therefore, the classification of autumn-sown wheat zones was originally based on sowing time. Our results show that in addition to winter cultivars, Zone II includes spring cultivars with a residual vernalization requirement (presence of Vrn-D1). We also showed that the majority of modern cultivars released in Zones III and IV have at least one dominant Vrn allele and should be classified as spring type. These results confirmed previous observations from Chinese breeders (Zhuang, 2003). We propose to rename Zone II as the Yellow and Huai River Valley Autumn-Sown Winter and Spring Wheat Zone and Zones III and IV as the Middle and Lower Yangtze Valley Autumn-Sown Spring Wheat Zone, and the Southwestern Autumn-Sown Spring Wheat Zone, respectively. This view is supported by He et al. (2001).

In conclusion, growth habits and distribution of dominant vernalization alleles among various wheat zones in China were significantly different. All cultivars released in Zone I were winter types and carried recessive alleles at the four vernalization loci. In Zone II, both winter and spring cultivars were present, and the latter usually carried a single dominant Vrn-D1 allele. In Zones III and IV, spring cultivars with the single dominant Vrn-D1 allele were frequent. In spring-sown Zones VI, VII, and VIII, all cultivars were spring, and most of them carried the strongest dominant vernalization gene Vrn-A1 plus other dominant gene(s). The distribution of growth habit and vernalization alleles in the different wheat zones of China were largely determined by the severity of the winter temperatures and the length of the growing season.

Appendix


   ACKNOWLEDGMENTS
 
The authors are grateful to Prof. Robert McIntosh from the University of Sydney for kindly reviewing this manuscript. Dr. Jorge Dubcovsky acknowledges support from USDA-NRI grant 2007-35301-17737. This study was supported by the National 863 Program (2006AA10Z1A7 and 2006AA100102) and the Ministry of Agriculture of China (2006-G2 and 2006BAD01A02).


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Received for publication September 6, 2007.


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