Control of Awn Length in Rice Breeding Programs in Hokkaido

Understanding genetic diversity is a primary goal of the molecular evolution of the genes. Awn length is a well-documented phenotype among domestication traits in rice, from long to short awn. In addition, awnlessness is favor for current rice farmers. Here, we identied the genetic basis of awn length during rice breeding programs in Hokkaido. We found variation of awn length ranging from 0.0 to 37.6 mm. Varieties with a short-awn or awnlessness have been selected under rice breeding programs. Genetic analysis on awn length identied that RAE1 and RAE2 on chromosomes 4 and 8, respectively, accounted for awning. These genes were well known to be signicant during Asian rice domestication. Sequence variations of the genes would clarify the molecular evolution of the genes on awn length. Firstly, the loss-of-function allele in RAE1, rae1, was selected for short awn length. Then, alleles on RAE2, RAE2-H01 to RAE2-H04, were targeted for the selection of short-awn or awnlessness. The selections on awnlessness phenotype could diversify these alleles on the genes, RAE1 and RAE2, exhibiting the variation of awn length.


Introduction
Understanding of the genetic diversity is a primary goal of the molecular evolution of the gene for sustainable plant breeding programs. Awn is a needle-like organ extending from the lemma tip of spikelet. Awns are characteristic of seeds of wild plants as they aid in seed dispersal (Elbaum et al. 2007; Guo and Schnurbusch 2016). They also contribute to the photosynthetic activity of the in orescence in wheat and barley, though not in rice (Rebetzke et al. 2016). Awn length of the short awn is a domestication trait in rice. Many genes for awn length have been characterized, some at the molecular those related to short awn length include Awn-1 (An-1), Awn-2 (An-2), LONG AND BARBED AWN 1 (LABA1), Regulator of Awn Elongation 1, 2, and 3 (RAE1, RAE2, RAE3), and Grain Length and Awn Development (GLA). Whereas awnlessness is one of the major targets for rice breeding programs. Awn could inhibit the handling of rice seed in rice cultivation. Awnlessness is favor for current rice farmers.
Asian cultivated rice, Oryza sativa L., is a major staple food that provides the caloric requirements for the world's population and originated in the tropics. Rice cultivation have increased rice production under various climatic conditions at latitudes between 53°N and 40°S (Lu and Chang 1980). In local rice areas, various kinds of traits might be successful under arti cial selection to establish rice cultivation. For example, those in Japan focus on eating quality and the adaptability to local environmental conditions (Fujino et al. 2019a;Kobayashi et al. 2018). Recent molecular genomics seek to understand the adaptability of historical processes in modern crop breeding programs. The genetic and phenotypic diversity on the traits has been characterized among varieties during rice breeding programs (Fujino et al. 2015(Fujino et al. , 2017(Fujino et al. , 2019aShinada et al. 2014). However, the genetic basis of the selections has not been clari ed.

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Dysfunctional alleles of RAE1 (on chromosome 4) and RAE2 (on chromosome 8), which are involved in the genes for the control of awn length have been selected during Asian rice domestication (Bessho-Uehara et al. 2016). Comparative analysis of RAE1 and RAE2 revealed that a two-step loss of function contributed to awn length (Bessho-Uehara et al. 2021). Loss-of-function alleles of both genes, rae1 and rae2, are typical in japonica rice (Bessho-Uehara et al. 2021). RAE1 encodes a bHLH transcription factor (Luo et al. 2013 This study focused on the variation of awn length on the transition from long awns to short awns or awnlessness during rice breeding programs in Hokkaido. Genetic analysis identi ed that RAE1 and RAE2 contribute to awn length including awnlessness. To clarify the selection history of awn length, we searched for the sequence variations of RAE1 and RAE2 and phenotype of awn length. We could elucidate the genetic base for the control of awn length in rice breeding programs in Hokkaido.

Materials And Methods
Plant materials 'Akage' and 'Kitaibuki' were used as parents for linkage analysis for awn length. 'Akage' is a long-awned landrace from Hokkaido, Japan, at one of the northern-limits of rice cultivation in the world (Fujino et al. 2017(Fujino et al. , 2019a. A rice variety 'Kitaibuki' is awnless and was registered in 1990. To identify QTLs controlling awn length, we developed an F 2 population (n = 181) derived from a cross between them. In brief, F 1 plants were self-pollinated to produce F 2 seeds.
We compared sequences of genes for awn length among varieties from three populations. One was the Differences between means were tested by two-way analysis of variance (ANOVA) and by Tukey-Kramer HSD test to show epistatic interactions between genes.

DNA analysis
Seeds from Genebank were sown for DNA isolation without propagation. Total DNA was isolated from young leaves by the CTAB method (Murray and Thompson 1980). PCR and sequencing were performed as described by Fujino et al. (2004Fujino et al. ( , 2005. Primers for genotyping of chromosomal regions for RAE1 and RAE2 in the F 2 population are listed in Supplementary Table 1. These indel markers were developed by the myINDEL procedure (Fujino et al. 2018).
We compared the RAE1 and RAE2 sequences. PCR experiments to test for the presence or absence of a transposon insertion in RAE1 were carried out using three primer pairs that target the transposon and the genomic regions anking the insertion of the transposon ( Supplementary Fig. 1, Supplementary Table 1). The transposon insertion causes loss-of-function of the gene (Luo et al. 2013).
The genomic region of RAE2, including the upstream region, coding region, and downstream region, was ampli ed and sequenced.
DNA sequences were initially aligned in BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and then adjusted by eye. All polymorphisms were rechecked from chromatograms with special attention to low-frequency polymorphisms. Heterozygosity was not observed.
Sequence data for haplotyping around RAE1 and RAE2 are drawn from the SRA/ENA/DRA databases under accession numbers DRA006061 and DRA008447.

Variation in awn length among HRCP cultivars
Awn length varied among varieties in HRCP, from 0.0 mm in 26 varieties, which were breeding lines, to 37.9 mm in 'Akage' (Fig. 2, Supplementary Table 2). Among seven landraces, it ranged from 0.0 mm in 'Bouzu' and 'Wasebouzu' to 37.9 mm in 'Akage' (Supplementary Table 2). Awns longer than 3.5 mm were not observed in any of the 23 varieties bred since 1975 (Supplementary Table 2). This phenotypic change on awn length might be derived from the genes for the trait during rice breeding programs.

Two chromosomal regions for awn length
To identify chromosomal regions controlling awn length, we carried out genetic analysis using an F 2 population derived from 'Akage' (39.9 ± 8.9 mm) × 'Kitaibuki' (0.0 ± 0.0 mm). Awn length in the F 2 population varied widely with a continuous distribution, from 0.0 to 54.9 mm (Fig. 3A). Next, to identify the genetic bases of this awn length regulation, association of the awn length was examined. It is known that RAE1 and RAE2 contribute for the control of awn length in Asian cultivated rice (Bessho-Uehara et al. 2016). A clear association between awn length and the genotypes of the marker AwnAKKT102 on chromosome 4 (QTL1) near to RAE1 and the marker AwnAKKT204 on chromosome 8 (QTL2) near to RAE2 (Furuta et al. 2015) was determined among the F2 population (Fig. 3, Supplementary Table 3). We concluded that RAE1 and RAE2 accounted for the awn length.
According to the genotype of two QTLs, QTL1 and QTL2 for RAE1 and RAE2, respectively, plants in this population were classi ed into nine genotype classes (Fig. 3B, Supplementary Table 3

Sequence variations in RAE1
Next, we identi ed sequence diversity in RAE1 and RAE2 for awn length. The transposon insertion in RAE1 caused loss-of-function allele, rae1 (Luo et al. 2013). We used the PCR procedure to survey the presence/absence of the transposon (Supplementary Fig. 1 Tables 4, 5). These results suggest that the functional allele conferring the awned phenotype is maintained among the populations, HL and JRC, which might provide a morphological marker for distinguishing varieties before rice breeding programs on scienti c theory (Table 1).

Sequence variations in RAE2
Next, sequences of RAE2 in HRCP were compared with the RAE2 reported in Bessho-Uehara et al. (2021). In addition to the 6-bp insertion in all HRCP varieties, we identi ed four polymorphisms ( Supplementary   Fig. 2; Supplementary Table 2). We could identify four alleles, RAE2-H01~RAE2-H04. RAE2-H01 had no polymorphisms. RAE2-H02 had a 6-bp deletion generating a 2-amino-acid deletion and a 1-bp polymorphism generating a 1-amino-acid substitution. RAE2-H03 had a 2-bp deletion generating a frameshift in translation. RAE2-H04 had a 4-bp deletion generating a frame-shift in translation. By the polymorphisms in the GC-rich region in RAE2, RAE2-H01, RAE2-H03, and RAE2-H04 identi ed in this study were identical to RAE2-hap 1, 5, and 3, respectively, of Bessho-Uehara et al. The selections of the RAE2 allele seemed to be along with the process of rice breeding programs ( Table  2). In the initial phase, there was RAE2-H01, which is functional allele. Then, RAE2-H02 and RAE2-H03 were selected and appeared. Finally, RAE2-H04, was detected at two varieties, 'Shimahikari' and 'Nanatsuboshi', which were bred in 1981 and 2001, respectively (Supplementary Table 2).
In the RAE2 region, we identi ed three major haplotypes, Hap=RAE2=H01 to Hap=RAE2=H03 (Fig. 4B, Supplementary Table 7). Hap=RAE2=H01 was divided into ve sub-haplotypes, Hap=RAE2=H01a to Hap=RAE2=H01e. Four alleles in RAE2 correlated well with haplogroups (Fig. 4B). Allele RAE2-H01 was present in Hap=RAE2=H01a, c, and e. RAE2-H02 was in Hap=RAE2=H01d. RAE2-H03 was in Hap=RAE2=H02. RAE2-H04 was in Hap=RAE2=H03. Here, we focused on the controlling of awn length among varieties in rice breeding programs in Hokkaido. Genetic analysis in this study revealed that two genes, RAE1 and RAE2, contribute to the controlling of awn length. Sequence variations in RAE1 and RAE2 revealed the diversi cation of alleles in the genes under the selection history of awn length during rice breeding programs. At rst, RAE1 was under the selection. The loss-of-function allele of RAE1 as the transposon insertion allele, rae1, was selected. This allele deduced awn length. Then, RAE2 was under the selection (Table 2), resulting in shorter awn length and awnlessness, rae1 rae2 (Table S2).

Discussion
Diversity in RAE1 and RAE2, which were already characterized as domestication genes (Bessho-Uehara et al. 2016), found in this study revealed the dynamics of molecular evolution of the gene. RAE1/An-1 was divided into two major haplotypes between cultivated rice and wild rice, and the haplotype in cultivated rice classi ed into two sub-haplotypes (Luo et al. 2013). Our results show that awned varieties carry RAE1 (without transposon insertion), whereas short-awned or awnless varieties carry rae1 (with transposon insertion). We found both two alleles, RAE1 and rae1, in a single local population. While we found four alleles at RAE2, RAE2-H01 to H04. Genes for awn length have pleiotropic effects on yield traits ( We here propose a model of molecular evolution in RAE1 (Fig. 5). Two haplotypes with a functional RAE1, Hap=RAE1=H01 and Hap=RAE1=H03 were ancestral. Transposon insertion into RAE1 in Hap=RAE1=H01 generated rae1. Then, rae1 might recruited into two haplotypes, Hap=RAE1=H02 and Hap=RAE1=H03. The introgressions of rae1 appear to have been generated from crossing-over of micro-chromosomal segments within a region of up to 7806 bp between haplotypes. Due to the crossing-over, we found ve combinations of RAE1 alleles with haplotypes around RAE1.  Tables   Table 1 to 3 are only available