Functional gene assessment of wheat: breeding implication in Ningxia province

16 Background: The overall genetic distribution and divergences for cloned genes 17 among wheat varieties that occurred during the breeding process of the past few 18 decades in Ningxia province of China are poorly understood. Here we report the 19 genetic diversities of 44 important genes underpinning grain yield, quality, adaptation 20 and resistance in 121 Ningxia and 86 introduced wheat cultivars and advanced lines. 21 Results: Population structure indicated characteristics of genetic components of 22 Ningxia wheats including landraces of particular genetic resources, introduced 23 varieties with rich genetic diversities and modern cultivars in different times. Analysis 24 of allele frequencies showed that dwarfing alleles Rht-B1b at Rht-B1 and Rht-D1b at 25 Rht-D1 , 1BL/1RS translocation, Hap-1 at GW2-6B and Hap-H at Sus2-2B are present 26 very frequently in Ningxia modern cultivars and introduced varieties from other regions, but absent in landraces, indicating that the introduced wheat germplasm with 28 numerous beneficial genes are vital for broadening genetic diversities of Ningxia 29 wheat varieties. Large population differentiation occurred at adaptation genes between 30 modern cultivars and well-adapted landraces. Founder parents have excellent allele 31 combinations of important genes with a higher number of favorable alleles compared 32 with modern cultivars. The gene flows manifested that six founder parents greatly 33 contributed to breeding improvement in Ningxia province, in particular Zhou 8425B 34 for yield related genes. 35 Conclusions: These results will greatly benefit for wheat breeding in Ningxia 36 province and other areas with similar ecological environments. 37

4 hybridization breeding [34] were cloned recently. The Lr34/Yr18/Pm38 locus 86 conferring durable adult-plant resistance to multiple diseases is used in wheat 87 breeding programs worldwide [30]. The 1BL/1RS translocation (1BL/1RS) has been 88 adopted widely in wheat breeding due to positive impacts on grain yield, adaptation, 89 and particularly the presence of resistance genes to several diseases and pests 90 although its translocation is associated with undesirable bread-making quality [35]. 91 Modern breeding has imposed selection for improved productivity that largely 92 influences the frequency of superior alleles for genetic loci underpinning traits of 93 breeding interest. Therefore, molecular diagnosis for the allelic variations of genes is 94 important to manipulate beneficial alleles in wheat molecular breeding. Enhanced 95 capacity in sequencing, along with the availability of high-quality genome sequences 96 of bread wheat, has allowed researchers to deploy specific favorable alleles using 97 molecular markers. Currently, 157 functional markers documented for more than 100 98 loci underpinning adaptability, resistance to biotic and abiotic stresses, quality and 99 grain yield were converted into high-throughput KASP assays [36]. Such approaches 100 will promote assessing the distribution of functional genes of wheat germplasms and 101 applications in wheat breeding. 102 Our objectives for this study were to evaluate genetic structure, diversity,   (Table S1). The latter 115 were introduced in Ningxia province over the past decades and played a huge role in    (Table S1). All selected KASP assays showed clear clustering results of 153 varieties ( Figure S1). In total, these loci underpin grain yield (10), quality (14), 154 adaptation (6), and stress resistance (14).

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The neighbor-joining analysis divided 207 varieties into two groups, designated 156 as Ningxia and Others, respectively ( Figure 1A), in agreement with PCA ( Figure 1B).

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The number of subpopulation (K) was plotted against the ΔK calculated from the 158 Structure, and the peak of the broken line graph was observed at K = 2 ( Figures 1C,   159 S2), demonstrating that the population was basically divided into two subgroups. The

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To further clarify the large genetic differences between germplasms from Ningxia and 169 Others, genetic diversities and variations were assessed. There was apparent 170 7 difference in genetic diversity at 44 loci controlling yield, quality, adaptation and 171 stress resistance between Ningxia wheat germplasms and Others (Figure 2A). Further 172 exploration indicated a higher genetic diversity at ten grain yield loci in the group 173 Others compared with Ningxia wheat varieties (P < 0.01), whereas Ningxia had a 174 higher genetic diversity than Others at 14 quality genes (P < 0.05) ( Figure S4A, B).

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Among them, genetic diversities estimated at Cwi-4A, GS-D1, Sus2-2B and Sus1-7B 176 loci for yield were abundant in the Others subgroup, while genes of Glu-B1, Glu-D1, 177 Pina-D1 and Zds-A1 for quality showed much higher genetic diversities in the 178 Ningxia subgroup relative to Others (Table S3). In addition, we also found that 179 genetic divergence was most obviously at quality genes, followed by yield genes 180 ( Figure 2B). An in-depth analysis showed an evident genetic divergence at some loci  (Table S3). To investigate 183 genetic divergences on gene level, we evaluated allele frequencies at genes for grain 184 yield, quality, adaptation and stress resistance as indicated below.   Ppo-A1b, Pod-A1b and Zds-A1a alleles, associated with lower PPO activity, higher 199 POD activity and lower yellow pigment content, respectively, were more frequent in

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The results showed that wheat accessions from Ningxia clustered in two clades, i.e. 228 landraces and modern cultivars, respectively. Therefore, we further analyzed genetic 229 9 relationship between landraces and modern cultivars. We found a higher genetic 230 diversity in modern cultivars compared with landraces ( Figure 3B). Moreover, the 231 difference of genetic diversities was clearly manifested in adaptation-related genes, 232 while no significant differences between two groups were observed in other genes 233 controlling yield, quality and resistance ( Figure S6). Population differentiation (Fst) 234 and gene frequency against the four types of genes were also analyzed to reveal 235 substantial divergences between two subgroups below.  (Table S4), whereas corresponding favorable allele frequencies had distinct 255 differences between modern cultivars and landraces (5% vs 54%, 5% vs 47%, 50% vs 256 92%, 67% vs 30%, 22% vs 0%) ( Figure S8).        Availability of data and materials 557 The datasets generated and analyzed during the current study are available from the 558 corresponding author on reasonable requests. 560 The authors declare no conflicts of interest.